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BIOLOGICAL STAINS 



A Handbook ox the Nature and Uses of the Dyes 
Employed ix the Biological Laboratory. 



By H. J. coxx 

New York Agricultural Experiment Station 
Chairman, CoxfMissiox ox Standardization of Biological Stains 

Prepared with the collaboration of: 

J. A. Ambler, S. L' Kornhauser, F. B. Mallory, and L. W. Sharp 

Members of the Executive Committee 
of the Commission 



Published by the Commission 

Geneva, X'^. Y. 
U. S. A. 

1925 



COPYRIGHT 1925 
Commission on Standardization of Biological Stains 



COMPOSED AND PRINTED BY 
W. F. HUMPHREY, 
GENEVA, N. Y., U.S. A 



TABLE OF CONTENTS 

PAGE 

Preface 5 

Chapter I. History of staining 7 

Chapter II. The general nature of dyes and their classification 11 

Chapter III. The spectrophotometric analysis of dyes 25 

Chapter IV. Dyes of the nitro, azo, and oxyquinone groups 32 

1 . The nitro group 32 

2. The azo group 33 

3. The oxyquinone group 42 

Chapter V. The quinone-imide dyes 44 

1. The indamins 44 

2. The thiazins 44 

3. The oxazins 51 

4. The azins 53 

a. Amido-azins or eurhodins 53 

b. Safranins 55 

5. The indulins 57 

Chapter VI. The phenyl methane dyes 58 

1. Di-phenyl methane derivatives 60 

2. Tri-phenyl methane derivatives 60 

a. Di-amino tri-phenyl methanes 61 

b. Tri-amino tri-phenyl methanes (rosanilins) 62 

c. Hydroxy tri-phenyl methanes (rosolic acids) 73 

Chapter VII. The xanthene dyes 75 

1. The pyronins 75 

2. The rhodamines 77 

3. Fluorane derivatives 78 

4. Phenolphthalein and the sulphonphthaleins 83 

Chapter VIII. Compound dyes 86 

Chapter IX. The natural dyes 91 

The indigo group 91 

Cochineal products 92 

Orcein and litmus 94 

Brazilin and haematoxylin 95 

Chapter X. The theory of staining . 98 

Appendix I. Tables relating to stains 105 

Appendix II. Commission specifications of certain stains 132 

Appendix III. Bibliography 138 



PREFACE 

WHEN microscopists first began, in the sixties and seventies, 
to use stains, the demand for dyes for this purpose was 
naturally too small to justify a special source of supply. 
They therefore had to make use of textile dyes, which were then 
very crude and were not constant in their composition. After a 
number of years, however, the demand for biological stains grew 
and a special commercial source of supply for them first appeared 
in Germany. This was the Grl'ibler Co., later Griibler and Hol- 
born. This company did not manufacture the dyes, as used com- 
monly to be thought in other countries; but on the other hand it 
cannot be denied that its founder made a distinct contribution to 
science in making the first effort to secure constancy and reliability 
in dyes intended solely for the use of the microscopist. It is sup- 
posed that he tested dyes under the microscope himself, and if a 
batch proved satisfactory in his experience bought a supply large 
enough for a number of years, bottled it under his own label and 
sold it to biologists. There is no question but that in this way the 
biologist was furnished with a much more reliable line of stains 
than if he had been obliged to buy directly from the dye manufac- 
turers; but it was an empirical method of standardization and there 
was nothing to prevent different batches of some dye secured by 
this company from varying considerably in their composition. 
Such upon investigation has proved to be the case. 

Altho a great service was done to biologists by this company in 
the latter part of the nineteenth century, such methods of stan- 
dardization are not in keeping with modern scientific knowledge. 
A recent cooperative undertaking has therefore been organized in 
America to put the standardization of stains upon a scientific basis. 
This undertaking started after the war had caused a shortage of 
stains, with the object of securing a reliable supply when the foreign 
sources were unavailable. It has since then been widened in its 
scope; and now that the foreign products are again available, the 
purpose of the work is to effect a scientific standardization of 
stains whether derived from foreign or domestic sources. As a 
matter of fact, so far only domestic samples have been considered. 
This has not been because of any prejudice against foreign stains, 
but because of practical difiiculties; it is, in brief, difficult to test 
each batch before it is put on the market when the concern handling 
it is in Europe. 

The organization thru which this work is being carried on is 
known as the Commission on Standardization of Biological Stains. 
It was organized in 1922 under the auspices of the National Re- 
search Council and is still affiliated with it, altho now no longer a 



part of the larger body. It is in effect a coordinating committee 
representing the American Chemical Society, the American Society 
of Bacteriologists, the Society of American Zoologists, the Botan- 
ical Society of America, the American Association of Pathologists 
and Bacteriologists and the American Association of Anatomists. 
It has a membership of about sixty biologists, members of the 
various societies just mentioned, who assist in the examination and 
testing of stains, each in those particular lines of technic with which 
he is especially familiar. It has secured the cooperation of chem- 
ists, dye manufacturers and stain dealers, so as to be sure that the 
needs of biologists can be immediately reflected in the supply of 
stains on the market. Its affairs are managed by an executive 
committee of five members, the present members of which repre- 
sent bacteriology, botany, dye chemistry, pathology, and zoology, 
respectively. This executive committee has undertaken the pre- 
paration of this book. The authorship of the book has been as- 
sumed by the chairman of the committee, however, in order to fix 
the responsibility and to make bibliographic references to it 
simpler than in the case of plural authorship; but the assistance of 
the other committee members in the work has been so great that 
they may be practically considered co-authors of the book. The 
chairman of the committee, therefore, wishes to take this occasion 
to acknowledge the invaluable assistance given by these other 
members. Without their cooperation such an undertaking would 
have been impossible. 

The chief object of the book is to present in logical form the in- 
formation which has been accumulating in the hands of the Com- 
mission since it was organized. It is neither a treatise on dye chem- 
istry nor one on microscopy; altho it contains information in both 
fields. It is an effort to present in a form acceptable to biologists 
the principles of dye chemistry so far as they have a bearing on 
biological stains; and to discuss the suitability of the different dyes 
for various biological purposes, presenting data partly original and 
partly drawn from the literature. The subject matter is realized to 
be incomplete, particularly that part of it which deals with the 
biological uses of dyes. An effort has been made to list the most 
important present uses of stains, and of the obsolete uses to men- 
tion those of historical significance; but it is realized that there must 
be many omissions. It is hoped that readers of the book will co- 
operate by calling to the author's attention places where the treat- 
ment of any subject seems inadequate. 

H. J. Conn, Chairman, 
Commission on Standardization 
of Biological Stains. 
Geneva, N. Y., 1925. 



6 



CHAPTER I 

HISTORY OF STAINING 

CONSIDERING how dependent microscopists are today upon 
the use of stains, it is hard to reahze that much important 
work had been done with the microscope before the use of 
stains was attempted. Altho natural dyes such as carmin and 
indigo were well known in the early days of the microscope, their 
use in staining microscopic preparations does not seem to have been 
mentioned until about 1850; and anilin dyes were not put on the 
market until 1856. Yet anyone who has studied the history of 
biology must realize that many discoveries had been made with 
the microscope before this period. 

It is safe to say, nevertheless, that the use of stains revolutionized 
microscopic technic. The early microscopists were able to make 
much progress without stains because of their painstaking dili- 
gence. The work without stains must have been extremely diflScult, 
and it is hard on reading some of the old publications to believe 
that some of the minute structures described were actually seen. 
Few users of the microscope today would be likely to have either 
the patience or the eyesight to do the work described in those early 
days. The fact that the microscope is now being used successfully 
in the hands of so many students who would not think of comparing 
themselves with the pioneers in microscopy is due to the use of 
stains more than to any other factor — altho of course no one can 
deny that modern improvements in the microscope have also 
played a part of great importance. 

The first use of a dye in microscopic work seems to have been by 
Ehrenberg (1838)*, who did not intentionally stain his specimens, 
but devised the scheme of grindmg indigo and carmin into a very 
fine powder and feeding them to the microorganisms he was study- 
ing. His idea in doing this was that the organisms would consume 
the dye bodily and that their digestive system could be traced by 
observing what portions of the body became colored. Ehrenberg 
found by this method that the organisms in question showed cer- 
tain zones or bands of color; and assuming that each of these 
colored spots was a stomach, he named the group of organisms 
"Polygastrica." Inasmuch as the group included bacteria and 
protozoa, the name was somewhat of a misnomer. It is interesting, 
however, to realize that Ehrenberg's technic is still used to demon- 
strate the ability of protozoa to engulf food particles. 

The early history of staining from this time on is given in a very 
interesting manner by Gierke (1884-5) and by Mann (1902), one 

*For references cited see Bibliography pp. 138 to 145. 



or the other of which sources is recommended to anyone desiring a 
more detailed account than is given here. 

Besides the fairly common application of iodine to microscopic 
preparations there seems to have been no further effort to color 
objects under the microscope until the middle of the century. At 
this time carmin was used almost simultaneously by two botanists, 
Coppert and Cohn (1849) and by a zoologist Corti (1851). Goppert 
and Cohn used the dye to assist them in studying the rotation of 
the cell contents of Nitella flexilis. Corti's work is overlooked by 
both Gierke and by Mann; but he states definitely (p. 143) that to 
observe distinctly the epithelial cells one should color them lightly 
with a solution of sugar or of carmin in a mixture of half water and 
half alcohol. This work was very promptly followed by another 
use of the same dye — the staining of chlorophyl granules in plants 
— by Hartig (1854 and 1858). In studying the nucleus of cells he 
made use not only of carmin but of litmus and black ink; and he 
observed that while albumin and gelatin were easily stained, the 
dyes had no action on such material as gums and mucin. Altho 
these authors did careful scientific work and certainly were the first 
users of dyes for histological purposes, their work apparently at- 
tracted no attention at the time. The real introduction of bio- 
logical stains was made by Gerlach (1858). 

Altho Gerlach did not discover the action of dyes on microscopic 
objects, nor was he the first to use carmin, nevertheless he should 
be, and generally is, considered the father of the technic of staining. 
Having observed that tissues became colored after injection with a 
poorly prepared carmin gelatin, he devised the scheme of preparing 
an ammoniacal carmin. He was therefore the first to use ammon- 
ium carminate, which is so nearly indispensible to modern histolo- 
gists. His early efforts with it, however, were unsuccessful — until 
he had a lucky accident which revealed the source of his trouble 
and opened up the way for further work. He happened to leave a 
section of nervous tissue, which had been hardened in potassium 
bichromate, over night in a very dilute carmin solution. When he 
examined it twenty -four hours later he found that it was beauti- 
fully stained, with fine differentiation of nerve fibers and nerve 
cells. His earlier failures had been due to the use of too strong a 
solution of the dye. This gave the key which helped solve the 
problem of tissue staining. Advances came quickly after that; for 
Gerlach had shown the way, and others merely had to follow. 

These advances were not wholly with carmin; altho at first the 
anilin dyes were scarcely kno"v\-n and the number of stains available 
to microscopists was quite limited. Indigo was first employed by 
Maschke (1859) who was familiar with Hartig's work but not with 
Gerlach's. It is stated (anonymous, 1865) that Thiersch and 
Miiller had just developed a technic employing carminates in com- 
bination with oxalic acid, and a year later Schweigger-Seidel and 

8 



Dogiel (1866) introduced a combination of carminates with acetic 
acid. Haematoxylin was first introduced as an histological stain 
by Bohmer (186.5), altho a previous rather unscientific attempt had 
been made by Waldeyer (1864) to stain axis-cylinders by means of 
the watery extract of logwood. Bohmer's greater success was due 
to his use of haematoxylin crystals in combination with alum, 
either by accident, or else because he knew that it was frequently 
used as a mordant in textile dyeing. Slightly later Frey (1868) 
showed that similar results could be obtained by mixing the mor- 
dant with the solution in which the tissues were fixed before they 
were stained. 

Double staining was introduced at about this same period, when 
Schwarz (1867) proposed fixing tissue in creosote and acetic acid, 
then staining 24 hours in very dilute ammonium carminate, and 
subsequently washing and staining for two hours in picric acid. A 
year later Ranvier (1868) first used a picro-carmin stain to obtain 
the same results by a single procedure. 

Anilin dves had become commercial articles before all these ad- 
vances with the natural dyes had been made, the first one having 
been introduced in 1856 when Perkin prepared mauveine. Fuch- 
sin, under the name of anilin red, appeared in 1858. The first sug- 
gestion of their use in histology seems to have been made by 
Beneke (1862), who used acetic acid colored with a lilac anilin, 
probably a mauveine or anilin violet; while two years later Wal- 
deyer (1864) used anilin red (fuchsin) and also a blue and a violet 
anilin dye. The latter investigator observed the ability of fuch- 
sin to stain nuclei more deeply than cytoplasm and the axis cylin- 
der more deeply than the medulary sheath of nerves. 

The principle of differentiation following staining was soon in- 
troduced. Bottcher (1869) differentiated his sections by partially 
decolorizing with alcohol after staining with rosanilin nitrate. A 
very similar method was later published by Hermann (1875), who 
is often mistakenly given the credit for originating the principle. 
Later the same procedure was further investigated by Flemming 
(1881) who tried both acid and basic dyes, finding that the method 
was satisfactory only in the case of the latter group. 

Gierke (1. c.) in his historical discussion of staining says that the 
history up to his day (1884) was divided into three periods, each 
occupying a decade. The first decade, the fifties, was character- 
ized by a few important but unrelated discoveries, which ended in 
the work of Gerlach, each investigator following up accidental 
observations on the staining powers of carmin and the other well- 
known dyes of those days. After Gerlach's work the development 
of the technic in the sixties was more rapid and depended less upon 
chance success by the individual investigator; the effort was made 
to use similarlv all the dves and metallic colors then available. The 
next decade would have had much less left to develop in this line if 
it had not been that by this time the great variety of anilin dyes 

9 



were available and microscopists were constantly finding new uses 
for them. Gierke wondered if there would be any opportunity for 
equal development during the ten years to follow his paper. 

That development did not stop in his day is well known. Scarcely 
a year has passed without the introduction of some new staining 
technic of considerable importance. Sometimes dyes hitherto un- 
known to the biologist have been shown to be valuable in bringing 
out some particular structure; at other times new combinations of 
dyes have proved of special value for other purposes; while by 
other investigators it has been shown that old methods, used with 
modern refinements of apparatus and technic, may bring out details 
not dreamed of by the early histologists. But the farther this work 
has progressed the more the microscopist has become dependent 
upon his supply houses to furnish him reliable stains, so carefully 
purchased or manufactured that each lot ordered could be counted 
upon to duplicate the last. 

The preface of this book describes briefly how a company was 
formed in Germany to meet the demand for dyes for staining pur- 
poses that developed during the last three decades of the nine- 
teenth century, and how the recent post-war conditions, together 
with the modern demand for a more scientific basis for the industry, 
led to the establishment in America of a Commission on Stan- 
dardization of Biological Stains. The work of the Commission is 
two-fold. First, by cooperation of various biologists and chemists 
it is planning to get together all the available information concern- 
ing the nature of dyes as related to their use for various purposes in 
microscopic technic ; secondly, by working with the manufacturers 
it is trying to see that the supply of stains available in America is 
of the highest possible qualit}^ as judged by their performance in 
actual laboratory use. 

The first of these purposes has been partly accomplished by a 
series of brief notes appearing in certain biological publications; and 
is now being more fully reaHzed by the publication of this book. 
The second object is being slowly brought about by the plan of 
certifying stains. Manufacturers of stains are being encouraged to 
submit to the Commission, for testing, samples of different batches 
of their various stains. These samples the Commission compares 
by chemical tests and by submitting them to several biologists 
skilled in different lines of miscoscopic technic. Then in the case 
of those samples which prove satisfactory, the Commission allows 
the company to sell the batch tested with a label on it issued by 
the Commission, bearing the statement: "Found satisfactory by 
Commission on Standardization of Biological Stains for purposes 
mentioned on main label. Use for other purposes not contra- 
indicated unless specifically so stated on said label." This certi- 
fication is issued only for the particular batch tested. The manu- 
facturer is furnished only with enough labels to last for that batch 
in the ordinary course of trade; and occasional tests of market 

10 



samples are made to see if any samples sold under the label differ 
from the one originally tested. In this way it is hoped before long 
to have all the stains on the American market from batches which 
have passed chemical tests and have also been tested and approved 
by biologists skilled in their use. 

When stains are put on the certification basis, specifications for 
them, based on the Commission's tests, are prepared and future 
lots of stain submitted for certification are in all cases expected to 
fulfill these specifications. So far as these specifications have al- 
ready been drawn up they are listed in the appendix of this book. 



CHAPTER II 

THE GENERAL NATURE OF DYES AND 
THEIR CLASSIFICATION 

DYES are generally classed in two groups, the natural and the 
artificial. The former class is now of relatively smaller im- 
portance from the standpoint of the manufacturer and the 
textile dyer; for the artificial dyes far outnumber them and the 
advancement of science is gradually making it possible to produce 
many of the formerly natural dyes by artificial means. It just 
happens that one or two natural dyes, the derivatives of cochineal 
and logTvood extract (see Chap. IX) are among the most valuable 
biological stains; but the natural dyes in general are so few in num- 
ber that they can be practically disregarded in considering the 
general chemical nature of dyes. 

Because the first artificial dyes were produced from anilin, all of 
this class are often called "anilin dyes," altho there are now a large 
number of them which bear no relation to this compound and are 
not derived from it. Therefore the term is now quite largely being 
replaced by the more correct expression "coal-tar dyes," since all 
of them are made by chemical transformations from one or more 
substances found in coal-tar. 

BENZENE 

All coal-tar dyes may be considered as derivatives of the hydro- 
carbon, benzene, CeHe, which is the mother substance of the very 
important aromatic series of organic compounds. It is an unusual 
chemical compound in many respects, and it will be well, in order 
to understand the structure of dyestuffs, to review briefly one 
theory of its structure which accounts for many of its properties. 
The molecule of benzene is composed of six carbon atoms combined 
with six hydrogen atoms in such a way that each hydrogen atom is 
identical in all its reactions with every other hydrogen atom in the 
molecule. Now a carbon atom is considered to have in all cases 
four valency bonds, that is it is capable of uniting chemically with 

11 



four atoms of hydrogen which has a valency of one. The simplest 
and best way of expressing these facts by a structural formula is 
shown in the figure: 

c c 

I D 

c c 

The double bonds in this ring cannot be considered as stationary; 
for, if they were, a compound in which two adjacent hydrogen 
atoms had been replaced by other elements or radicals should 
occur in two different forms according to whether there were a 
single or a double bond between the two carbon atoms to which the 
substituting elements or groups were attached — which never proves 
to be the case. Hence the double bonds must be considered as 
mobile, each pair continually oscillating back and forth between 
the carbon atom bearing it and the two adjoining carbon atoms. 
In practice the formula for benzene is abbreviated to a simple 
hexagon : 




in which each corner represents a carbon atom. If no chemical 
symbol is placed outside the ring at any corner, it is understood 
that an atom of hydrogen is attached at that point. This configura- 
tion is spoken of as the "benzene ring." When the symbol of some 
element or radical is written at a corner, it means that the hydrogen 
atom at that point has been replaced by the element or radical to 
which the symbol refers. 

When two hydrogen atoms are replaced there are only three 
possible positions in the molecule which the replacing groups, or 
substituents, can take, as shown by the following figures, using 
chlorine as the substituent: 



CI CI 

Cf 





CI 



In the first formula, the substituents are said to be in the "ortho" 
position to each other; in the second they are in the "meta" posi- 
tion, and in the third in the "para" position. These three com- 
pounds are called respectively: ortho-dichlorobenzene, meta- 
dichlorobenzene, and para-dichlorobenzene. The three prefixes are 
commonly shortened to the respective initials: "o-" "m-," and 

12 



**p-." When the compound is complex it is customary to number 
the corners of the ring thus : 




In naming a compound in this way, the number of the corner to 
which a group is attached is given immediately before the name of 
the group. Thus, the three compounds shown above may be 
called respectively: 1, 2-dichlorobenzene, 1, 3-dichlorobenzene and 
1, 4-dichlorobenzene. 

There is another type of substitution in the benzene ring which 
is very important in dye chemistry. Two atoms or groups having 
two valency bonds instead of one may also replace two hydrogen 
atoms, provided the replacement takes place simultaneously and 
the hydrogen atoms replaced are situated either in the ortho or in 
the para position to each other. Thus two oxygen atoms (which 
are bivalent) may replace two hydrogen atoms (which are mono- 
valent) forming the compound known as quinone CeHA, the 
formula for which is 



or as commonly written 





M 

C C 

U I 

c C 








In printed formulae, such as those that follow in this book, the 
quinone ring is often abreviated still further by omitting the double 
bonds within the ring. The substituent atoms or groups may or 
may not be alike, so long as both have two valency bonds entering 
into the combination. This type of substitution involves a rear- 
ranging of the double valency bonds in the benzene ring; and in 
compounds of this type, called quinoid compounds, the double 
bonds are supposed to be fixed, not mobile as in benzene. This 
change of the valency bonds takes place very readily in many 
dyes, and certain peculiarities of their behavior are explained by 
it; (see for example p. 84). 

Three mono-substitution products of benzene are of importance 
in considering the structure of dyes, namely; toluene or methyl- 



13 



benzene, CeHo- CH3 ; phenol, carbolic acid or phenylic acid,C6H5- OH ; 
and anilin or phenyl amine, C6H5NH2. Their constitutional for- 
mulae are as follows: 

CH, OH NH. 

I ■ I I 

A A 

\/ \/ \/ 

toluene phenol anilin 

Two important di-substitution products are xylene or dimethyl 
benzene C6H4- (CH3)2, and toluidine, C6H4- CHs- NH2. Both of these 
occur in the above mentioned three isomeric forms, as shown below 
for xylene : 

CH3 CH3 CH3 

CH, I 1 





\/ \/\ \/ 



CH3 



\ 



CH, 



ortho-xylene meta-xylene para-xylene 

CH3 



NH 



2 




ortho-toluidine 

CHROMOPHORES, CHROMOGENS, AND AUXOCHROMES 

Certain groups of elements are known as chromophores because 
when they occur in a benzene derivative they impart to the com- 
pound the property of color. The benzene compounds containing 
chromophore radicals are known as chromogens. A chromogen, 
however, altho it is colored, is not a dye, in that it possesses no 
affinity for fibers or tissues. It may coat them, but only mechan- 
ically, and it will be easily removed by mechanical processes. 
That is, it will not "take." (See, however, the discussion of fat 
stains, p. 33). In order for a substance to be a dye, it must contain 
in addition to the chromophore group, a group which imparts to 
the compound the property of electrolytic dissociation. Such 
auxiliary groups are known as auxochromes. They may slightly 
alter the shade of the dye, but are not the cause of the color. Their 
function is to furnish salt-forming properties to the compound. 
Certain chromophoric groups have also slight auxochromatic 
properties. 

To illustrate these different types of groups, let us take a typical 
example. The nitro group (-NO2) is a chromophore. When three 

14 



of these groups displace three hydrogen atoms in a benzene mole- 
cule, we have the compound trinitrobenzene, 

0,X NO, 



NO. 

which is yellow. It is not a dye, however, but is a chromogen. It 
is insoluble in water, and is neither an acid nor a base; that is, it 
does not dissociate electrolvticallv and consequentlv cannot form 
salts with either alkalies or acids. If, however, one more hydrogen 
atom is replaced, this time with the hydroxyl group (-0H), which 
is an auxochrome, the resulting compound, 

OH 

O.X I NO, 




\/ 



NO. 

is an acid, capable of electrolytic dissociation and of forming salts 
with alkalies. It is the familiar substance picric acid, and is a 
yellow dye. 

It will thus be seen that the color of picric acid is due to the 
chromophoric nitro groups, and that its dyeing properties are due 
to the auxochromic hydroxyl group. If the nitro groups be reduced 
to amino groups (-XII2), which are not chromophores, the resulting 
compound is colorless and hence is not a dye. 

Summing up, we arrive at the definition of a dye as an organic 
compound which contains chromophoric and auxochromic groups 
attached to benzene rings, the color being attributable to the chro- 
mophores and the dyeing property to the salt-forming auxochromes. 

Some auxochromes are basic, e.g., the amino group (-XH2), 
while others are acidic, e.g., the hydroxyl group (-0H). The 
amino group owes its basic character (which it transmits to the 
whole molecule) to the ability of its nitrogen atom to become pen- 
tavalent by the addition of the elements of water (or of an acid), 
just as in the case of ammonia; thus: 

H H H H H H H 

/ \ \ / / \ / 

H— N + O = H— N and H— N +H— CI = H— N 

\ / / \ \ / \ 

H H H OH H H CI 

ammonia "water ammonium ammonia Hydro- ammonium 

hydroxide chloric chloride" 

acid gas 

15 



H H H H H H H 

/ \ \ / / \ / 

R— N + O = R— N and R— N +H— CI = R— N 

. \ / / \ \ / \ 

H H H OH H H CI 

amine water hypothetical amine hydro- amine 

organic am- chloric hy dro- 

mon ium base acid gas chloride 

The hydroxy! group, on the other hand, is weakly acidic, as it can 
furnish hydrogen ions by electrolytic dissociation. The more of 
either one of these two groups in a compound, the stronger base or 
acid it becomes. If there is one of each, the basic character of the 
amino group predominates, but is weakened by the influence of the 
acidic hydroxyl group. The strength of both groups is also influ- 
enced by other groups or atoms in the compound; thus, for ex- 
ample, the chromophore -NO2, altho incapable in itself of conferring 
acid properties to the compound, exerts an influence to make any 
hydroxyl group in the compound more strongly acidic, in other 
words to become more highly dissociated electrolytically. 

One other group of atoms encountered in dye chemistry needs 
explanation, namely the sulfonic group, -SO3H. It is a salt-forming 
group of strongly acidic character, in that it suffers extensive elec- 
trolytic dissociation . This group, however, is only very feebly auxo- 
chromic. Its function is to render a dye soluble in water, or to 
change an otherwise basic dye into an acidic one, as in the case of 
the fuchsins, where the strongly basic "fuchsins" are changed into 
the strongly acid "acid fuchsins" merely by the introduction of 
sulfonic groups into the former. A compound which contains a 
chromophore group and a sulfonic group is not a dye, however, 
unless there is also present a true auxochrome group. 

From what has been said above, it is not to be presumed that 
the dyes of commerce are actually bases or acids. Generally the 
basic dyes are sold as salts of a colorless acid, such as hydrochloric, 
sulfuric, oxalic or acetic acid. Likewise the acid dyes are sold as 
their sodium, potassium, calcium or ammonium salts. Occasion- 
ally the basic dyes are sold as the free bases, as for example the oil 
soluble dyes (see p. 38). When a basic dye which is ordinarily sold 
in the form of a salt comes into commerce as the free base, it is 
customary to use the word "base" immediately after the name of 
the dye. Thus, "basic fuschin" indicates a salt of fuchsin with a 
colorless acid, while "fuchsin, base" indicates fuchsin itself, not 
combined with an acid. 

THE CHROMOPHORES 

As stated above, every dye contains at least one group of atoms 
known as a chromophore, which is regarded as being responsible 
for the colored properties of the compounds in which it occurs. 
Some of these chromophores have a basic character, others acid. 
There are only a comparatively small number of them which enter 

16 



into the usual biological stains, and only these need be considered 
here. They are as follows : 



BASIC CHROMOPHORES 



1. The azo group, — X = N — , which is found in all azo dyes, of 
which methyl orange and Bismarck brown are well known ex- 
amples. In all these dyes, a benzene ring is attached to each 
nitrogen atom. All the dyes of this group may be looked upon as 
derivatives of azobenzene, 



X = X 





\/ 



2. The azin group. 



N 

/j\ 

\l/ 

N 

which is found in phenazins. of which neutral red and the safranins 
are good representatives. The skeleton formula of a safranin is: 

H,X X NHa 



X 

/ \ 



in which x represents the negative ion of a monobasic acid such as 
hydrochloric, acetic, nitric or sulfuric. This chromophore is 
capable of variety of rearrangements of its valency bonds, as the 
bond between the two nitrogen atoms may disappear and the com- 
pound assume a quinoid structure, as for example the following 
grouping: 



H.X /\ X /\_XH 




3. The indamin group, — X = , as observed in the indamins, 
thiazins, and so forth. Methylene blue is the best known repre- 
sentative of this group. In these dyes, two benzene rings are at- 



17 



tached to the nitrogen atom, one of these being in the quinoid form 
and hence adding a second chromophore. The typical indamin 
formula is: 



HN_/~\_N /"~\ NH 




In the thiazins, such as methylene blue, the two benzene rings are 
further joined together by a sulfur atom, forming three closed 
rings of atoms. The simplest thiazin base would be: 




_S_ /\_NH 
ACID CHROMOPHORES 



1. The nitro group, — NO2, as in picric acid. 

2. The quinoid benzene ring. 



which occurs in a long series of dyes, such as the indamins above 
mentioned, the xanthenes and the di- and tri-phenyl methanes, 
which include many well known stains, such as rosolic acid, 
fuchsin, methyl green and the methyl violets. A typical triphenyl 
methane formula is that of pararosanilin, base : 



C^/~"\^NH 





LEUCO COMPOUNDS 

The different chromophores differ considerably from one another, 
but they all have one property in common. In the language of 
chemistry, they all have unsatisfied affinities for hydrogen; or in 
other words, they are all easily reduceable, for combining with 
hydrogen is the opposite of oxidation and is, therefore, reduction. 
The nitro group may be reduced to an amino group; in the azin 
group the bond between the nitrogen atoms may break and two 
hydrogen atoms be taken on; while in the various chromophores 
with double bonds (such as the quinoid ring) the double bond may 
break and hydrogen atoms become attached to the valences thus 
freed. 

Now in every case this reduction destroys the chromophore 
group, and as a result the compound loses its color. In other words 
a dye retains its color only as long as its affinities for hydrogen are 

18 



not completely satisfied. These colorless compounds are known 
as leuco compounds; thus fuchsin yields leuco-fuchsin on reduc- 
tion, and methylene blue reduces to leuco-methylene-blue. For 
example: 




CH, 



_NH.C1+H.0 = H,N_ 



H,N_/ 



fuchsin 




XH3CI 



+0 



XH. 



leuco-fuchsin 



Ordinarily this reaction is reversable under conditions favoring 
oxidation. It is of especial significance to the bacteriologist, as 
dj^es can often be used as indicators of reduction. 

Certain dyes form a still different type of leuco compound, often 
called a "leuco-base." We have seen that the basic dyes ordinarily 
occur as salts of some colorless acid; now, in the case of certain 
dyes, notably the tri-phenyl methanes and xanthenes (Chapters 
VI and VII), as soon as the acid radical is removed, the compound 
becomes colorless. This is because a rearrangement of the atoms 
in the molecule takes place upon neutralization so as to give, not 
the true dye base, but a compound known as a carbinol (see p. 59) 
in which the chromophore does not occur. Thus the theoretical 
base of fuchsin which should be obtained upon removal of the acid 
radical is: 



CH3 

\ 
H.N_/ 



H.N_/- 



C=/ 




NH.OH 



The compound actually formed, however, is the pseudo-base or 
carbinol : 




H,N 



In this compound, it will be readily seen, there is no chromophore; 
hence it is colorless. These pseudo-bases are of little significance to 
the biologist, but they are of importance to the dye manufacturer 
as intermediates in the preparation of dyes. 

In the case of many acid dyes the chromophore is similarly 
broken bj^ a rearrangement of the atoms which occurs on neutrali- 

19 



zation. This reaction is ordinarily very readily reversable and 
makes such dyes useful indicators of acidity. It is discussed more 
fully under acid fuchsin (p. 64) and phenolphthalein (p. 83). 

CLASSIFICATION OF DYES 

On the basis of the chromophore present the simple synthetic 
dyes are classified into several groups. If each of these groups were 
characterized by a single color or by a few closely related colors, 
dye chemistry would be a comparatively simple proposition. As a 
matter of fact a single chromophore may occur in dyes of practi- 
cally all colors of the rainbow. It is ordinarily impossible to deter- 
mine, a priori, from the chemical formula of a dye what particular 
color the compound may have; but there is, nevertheless, a certain 
general rule which correlates chemical formula with color. In any 
group of compounds, the simpler ones are converted into the more 
complex by substitution of radicals for hydrogen atoms. In the 
dyes the substituents are generally methyl or ethyl groups, or 
sometimes phenyl groups. Now the general rule is that the larger 
the number of hydrogen atoms that have been replaced by these 
groups the deeper the color. The tendency is for the color of the 
simplest dyes in any group of homologous compounds to be yellow, 
passing thru red to violets and then greens and blues, as the homo- 
logs become higher thru the introduction of successively larger 
numbers of methyl or other substituting groups. Thus the com- 
pound pararosanilin, which is very frequently sold as basic 
fuchsin, but should more properly be called basic rubin, is a tri- 
phenyl methane, with an amino group attached to each benzene 
ring, but without any methyl groups; thus: 

H 

\ 

CI 

Rosanilin, which is similar in composition, but contains one methyl 
group attached to one of the benzene rings, 





is a red very similar to pararosanilin but with less of a yellowish 
cast. Now another methyl group may be introduced into each of the 
other two benzene rings, and each one successively deepens the 
shade of red, so that thehighesthomologof the series, new fuchsin: 



20 




has a more bluish cast than any of the others. Thus basic fuchsins 
can vary considerably in their shade according to the proportions 
in which these four possible components may be mixed. 

It is also possible in another way to deepen the color of pararo- 
sanilin still further, namely by introducing methyl groups into the 
amino radicals instead of directly on the benzene rings. Thus the 
methyl violets are obtained; and the more methyl groups intro- 
duced the bluer the violet, until when all six available hydrogen 
atoms are thus substituted, crystal violet, the deepest of them all, 
is obtained. By using three ethyl groups instead of methyl, 
Hoffman violet or dahlia is formed, which is deeper in color than 
the trimethyl compound, due to the heavier groups introduced. If 
three phenyl groups (i.e., the benzene ring (CeHs-) are introduced 
instead of methyl or ethyl, the color is still further deepened, the 
resulting dye being spirit blue. Further, it is possible to introduce 
another methyl group into crystal violet, by addition of methyl 
iodide (or chloride) to one of the trivalent nitrogen atoms, whereby 
its valency is increased to five, and a green dye, methyl green, is 
produced : 



CHj 


/ 


\ 


/ 


CH3-N / 


\ c 


/ \ 


. / \ 


CI 


\ 





With these facts in mind it will be seen that the grouping of dyes 
as based upon these chromophores does not classify them in rela- 
tion to their color. It is a useful classification, however, because it 
puts together those that have similar chemical structure. The 
important biological dyes, thus classified, fall into the following 
groups : 

1. The nitro dves. 

e.g., picric acid. 

2. The azo group. 

e.g., methyl orange, Bismark broicUy orange G, Congo red, 
Sudan III and Sudan IV. 

21 



3. The oxyquinone group. 

e.g., alizarin 

4. The quinone-imide group, including 

(a) Indamins 

(b) Thiazins; e.g., ihionin, toluidine blue, methylene blue. 

(c) Oxazins; e.g., brilliant cresyl blue, Xile bine. 

(d) Azins, including 

(i) Amido-azins; e.g., neutral red. 

(ii) Safranins; e.g., safranin 0, inadgala red. 

(iii) Indulins; e.g., nigrosin. 

5. The phenyl-methane dyes, including 

(a) diphenyl-methanes, e.g., auramin. 

(b) Diamino tri-phenyl methanes; e.g., malachite green, 
brilliant green, light green. 

(c) Triamino tri-phenyl methanes; e.g., basic fuchsin, acid 
fuchsin, methyl violet, gentian violet, methyl green, anilifi 
blue. 

(d) Hydroxy tri-phenyl methanes (Rosolic acids); e.g., 
aiirin, corallin red. 

6. The xanthene dyes, including 

(a) Pyronins; e.g., pyronin G and B. 

(b) Rhodamines; e.g., Rhodamine B. 

(c) Fluorane derivatives; e.g., eosins, erythrosin, rose 
bengal. 

(d) Phenolphthalein and the sulphonphthaleins. 

DYE NOMEXCLATURE 

Very little system has been used in naming dyes, and as a result 
their nomenclature is extremely confused. Generally the manu- 
facturer of a dye which he thinks is new or which he wishes the 
public to consider a new dye sells it under a new name which is not 
intended to give any clue as to the nature of the dye. If the 
manufacturer knows that the name is a mere synonym of one al- 
ready in use he does not say so, for he wishes to encourage the sale 
of his own product rather than that of some other dye maker. 
Accordingly it has been left for others, who are not financially 
interested, to work out the synonymy of the dyes; and the list of 
names that are found to apply to a single dye is sometimes amaz- 

With the dyes in general so unsystematically named, it is natural 
that the same confusion should reign in the nomenclature of bi- 
ological stains. This confusion is very unfortunate, for it often 
misleads the biologist as to just what he is doing. For example, 
some histologist may have on hand a bottle of stain labeled dahlia 
and he may find it useful for some new technic, which he publishes; 
while another may propose for an entirely different technic the 
stain Hoffman violet. Then a third laboratory worker may read 
both articles and wish to try both methods; so he accordingly 

22 



orders both dahlia and Hoffman violet. His dealer, who is prob- 
ably quite unacquainted with dyes, will very likely send him a 
bottle bearing each name, and the purchaser has no easy way of 
discovering that the two are identical; so he may continue for years 
to use the two stains for different purposes, misled by their labels 
and thinking them distinct. The manufacturers and dealers in 
stains have sometimes encouraged this confusion by their practice 
of taking care to have the label on the bottle agree with the name 
used in the customer's order, regardless as to what the usual name 
for the dve mav be. 

An attempt to relieve this confusion has been made by the Com- 
mission on Standardization of Biological Stains (1923f) by publish- 
ing a list of biological stains with their best known synonyms. In 
each case one of the names is listed as a preferred designation. 
Sometimes general usage made it easy to select one name as the 
preferred one; but in other instances the selection was more or less 
arbitrary. This same list, with a few revisions in the way of ad- 
ditions and corrections, is given in the appendix of this book (p. 
106). The preferred designations in this list are the same as in the 
earlier, except in the one case of methylene azure. For this stain 
Azure I was preferred in the earlier list; but as it was merely a 
trade name of somewhat uncertain application methylene azure 
seems preferable. The list of synonyms has been revised more 
extensively, largely to omit names that are obsolete and have no 
present meaning. 

DYE INDEXES 

Inasmuch as the dye industry originated in Germany and until 
the war was almost a monopoly of that country, it is natural that 
the first serious efforts to index the dyes should have been under- 
taken in that country. Until recently the only important index of 
dyes was Schultz's Farbstofftabellen, which is now in its sixth 
edition (1923). This index lists all of the important textile dyes, 
giving their synonymy, their chemical composition, methods of 
preparation, and distinctive characteristics. As these descriptions 
are concise, it seemed well to refer to the Schultz number of all the 
stains listed in the article on stain nomenclature above mentioned 
(Commission, 1923f), wherever such could be given. 

More recently another dye index has been published in England 
by the Society of Dyers and Colourists (1923). This publication, 
known as the Colour Index, is more complete than even the sixth 
edition of Schultz, and lists even such dyes as narcein, thionin and 
iodine green, which are no longer of use in the textile industry and 
have been omitted from recent editions of Schultz. The synonymy 
is more complete and up-to-date than that in Schultz, and many 
more chemical formulae are given. Accordingly in the following 
pages the stains are denoted by their Colour Index number (ab- 
breviated C. I. No.) instead of by their Schultz number, as in the 

23 



list previously published. The Schultz number of each of them is 
given for reference purposes, however, in the list in the appendix, 
p. 106. 

DYE SOLUBILITIES 

Textile dyes are never of a high degree of purity. Some of the 
impurities are accidental; others are added intentionally so that 
dyers can obtain the desired shade without having to measure out 
dyes in very small quantities. Inasmuch as the early biological 
stains were textile dyes without much, if any, modification, it is 
natural that some of them should also have been of low dye con- 
tent, and also that different batches should have been of various 
degrees of purity. In general the post-war dyes are much more 
pure thail those available before the war. This makes it difficult 
to prepare stain solutions identical in strength with those prepared 
before the war. 

There are two general types of stain formulae: in one a definite 
weight of dry dye is specified; in the other a certain volume of a 
saturated (generally alcoholic) solution of the dye. Each type of 
formula has its own possibilities of error; and to appreciate the 
problem it is necessary to understand certain facts in regard to the 
solubilities of dves. 

The error inherent in the first type of formula is plain at a 
glance. If two different staining solutions are made up containing 
1 g. per 100 cc. of dry methylene blue, and in one case the actual 
dye content of the dry stain is 90 percent, while in the other only 
55 percent (a difference actually observed in samples on the mar- 
ket), it is plain that the two solutions must differ greatly in their 
strength. For this reason an early recommendation of the Com- 
mission (1923b) was that formulae of the second type be preferred, 
on the assumption that a saturated solution of a dye would be more 
likely to be of constant dye content than different lots of dry stain 
bought in the market. 

This recommendation, however, was made without complete 
understanding of the actual facts of the case. The amount of a dye 
that will go into solution in either water or alcohol depends upon 
the amount of mineral salts present. If a dye contains a large per- 
centage of sodium chloride, for instance, a saturated solution will 
be of considerably lower actual dye content than if the dye were 
free or nearly free from salt; the sodium chloride prevents the 
solvent from taking up as much of the dye as it would normally. 
For this reason two staining solutions each containing 10 percent 
by volume of a saturated solution of the two methylene blues above 
mentioned would be quite different from each other in actual dye 
content, altho possibly more nearly alike than if they had been pre- 
pared with identical weights of the dry stain. 

As soon as these facts were fully understood, the Commission 
(1923e) modified its recommendation. It is plain that the only way 

24 



two staining solutions can be made identical if different batches of 
stain are used is to make them up on the basis of the weight of 
actual dye present in the stain used. This can be done only if the 
manufacturer has co-operated to the extent of printing the actual 
dye content of each batch of stain on the container in which it is 
sold. This is not yet commonly done; but the Commission is 
issuing its certification only to batches of stain on which the total 
dye content is stated. In this way it is hoped that eventually all 
stains on the market will be so labeled; and then when staining 
formulae are readjusted so as to call for definite quantities of actual 
dye, the preparation of staining solutions will be put on a more 
scientific basis. 



CHAPTER III 

THE SPECTROPHOTOMETRIC ANALYSIS OF DYES. 

DYES are extremely difficult to analyze by chemical methods. 
Their chemistry is in many cases obscure, they often differ 
from one another only in the way the chemical groups are 
combined together; different dyes may react alike to all known 
chemical tests and differ so slightly in solubility that it is difficult 
to distinguish one from another. For all these reasons it proves 
that spectrophotometric methods offer decided advantages in the 
examination of dyes over methods of chemical analysis, both in 
respect to general utility and in regard to convenience of appli- 
cation. 

When a ray of light passes thru a prism it is resolved, as is well 
known, into many rays differing from each other in wave length and 
in color. Now the color of any substance arises from the selective 
absorption or reflection of definite parts of the visible spectrum, as 
light passes thru or is reflected from this substance. In the visible 
spectrum is included light of wave lengths intermediate between 
about 400 and 725 millimicrons. (The millimicron, denoted by the 
symbol ijlijl, is one millionth of a millimeter in length.) The color 
of the light in the spectrum varies, with increasing wave length, 
from violet to red, appearing blue at about 450^ijLt, green at about 
500/i^i, yellow at about 550/x/x, and orange at about 600/xju, as shown 
in Fig. 1. The color of light which reaches the eye after trans- 
mission thru or reflection from a colored substance is comple- 
mentary to the color of the light absorbed by that substance. A 
violet dye, for example, appears violet because of its predominent 
absorption of yellow light. The complementary colors correspond- 
ing to the various parts of the spectrum are also shown in Fig. 1 
beneath the colors of the spectrum. 

The color of substances is ordinarily of complex origin, depending 

25 



upon the absorption of light in varying degrees, over an extensive 
spectral range. Whereas the unaided eye is able to register only 
the composite effect, it is possible to resolve this effect into its 
component factors with the aid of a spectrophotometer. Althothe 
eye is unable to distinguish between a violet dye and a suitable 
mixture of a red and a blue dye, the heterogeneous character of the 
mixture is readily apparent upon spectrophotometric examination. 
Pure dyes may have simple absorption spectra, in that their light 
absorption is all at one part of the spectrum, or they may be more 
complex, showing two or more points on the spectrum at each of 
which light is absorbed to greater extent than on either side of it. 
Thus even in the instance of pure products of identical color to the 
eye, the spectrophotometer frequently reveals decided differences 
when the character of the light absorption is considered in detail. 
The essential principle of spectrophotometric analysis may be 
understood by reference to Fig. 2, which is a diagram of a spectro- 
photometer. Two parallel beams of light of equal intensity enter 



Diagram of Spectrum 
Showing; complementary colors 



Wave lenatk: ^°° <" 500 550 600 650 ^^ 

*^ ' ' L 



> I 



Col or: violet blue green yellow orange re d 

Complementary color! yellow orange red violet bh 



lue 



Fig. 1. Diagram of spectrum 

the photometer box by separate orifices, pass thru a prism where 
they are resolved into visible spectra, and then reach the eye in 
contiguous fields so that very accurate comparison between the 
two spectra is possible. The arrangement is such that one beam 
passes directly to the prism whereas the intensity of the second 
beam may be reduced in any desired proportion by revolving the 
photometer circle. , A glass cell containing a dilute solution of the 
dye to be exainined is interposed in the path of the first beam and a 
similar cell containing water (or whatever solvent is used in the 
case of the dye) in the path of the second beam. The spectrum of 
the beam which has passed thru the dye solution will be found 
deficient in those portions which have been absorbed by the dye: 
and the degree of the deficiency at any position in the spectrum 
may be measured by determining the degree to which the intensity 
of the light of the second spectrum must be reduced in order to 
obtain an equal intensity in the two fields observed by the eye. 

,26 



The shutter of the eyepiece may be partially closed so that only 
a narrow spectral range is visible; this allows the eye to concentrate 
on the matching of two small fields, each of which appears uniform 
in color. The instrument is provided with a screw drum, calibrated 
in wave lengths, by means of which the prism may be rotated in 
such a manner as to bring light of any desired wave length into 
the center of the field of vision. 



irism 



divided circle for 
pnotometrit adjusW-nt 




-wave-length drum 
ior rotation of pnsm 



tye p. 



Diagram of 

Spectrophotometer 




water cell- 



photomaUr Lox 



.dy« eeD 



Parallel Yearns from 
common light source 

Fig. 2. Diagramatic section thru a spectrophotometer. 

The photometer circle may be calibrated in various ways. In 
the general examination of dyes it is convenient to obtain the data 
in the terms of a factor known as the Bunsen extinction coefficient 
(E), and to employ a circle from which such values may be read 
directly. 

In measuring the complete visible absorption of a dye, a series 
of measurements is made over the portion of the spectrum in 
which any appreciable absorption may be noted. This is done by 
setting the drum at some definite wave length and observing 
whether both beams of light reaching the eye are of the same in- 
tensity; if not, the photometer circle is turned until the beam 

27 



1^ . 




1.0 • 



420 



460 



500 



540 



580 



620 



eco 



Fig. 3. Absorption curves of five dyes of different colors: 

1. Tartrazine (yellow) 

2. Orange G. 

3. Fuchsin (red) 

4. Crystal violet 

5. Neptune blue BG. 



28 



which has not passed thru the dye is of the same intensity as that 
which has passed thru the dye cell. A reading of the extinction 
coefficient is then made. Further readings may be made at inter- 
vals of 10 fjLfjL, with intermediate determinations in the immediate 
vicinity of the maximum absorption or at any other point at 



IS 



1.15 



to.. 



75 



.5 ' 



.25 




42.0 



4£0 



500 



540 



5&Q 



6Z0 



ebo ><>* 



Fig. 4. Absorption curve of victoria green. 

vhich it may appear desirable to bring out detail. If extinction 
coefficients are then plotted against wave length a graphic repre- 
lentation of the absorption band of the dye is obtained. 

If measurements are carried out under suitable standardized 
conditions, the spectral position and the general form of the ab- 



29 



sorption curve are characteristic of the individual dye, while the 
magnitude of extinction coefficients (the height of the curve) varies 
directly with the amount of dye present. The absorption curves 
of dyes which are very closely related in structure are sometimes so 
similar as to be practically identical. In such instances the indi- 
vidual dyes may be recognized by means of quantitative deter- 
minations of the degree in which their absorption is modified under 
the influence of suitable variations in conditions. 



i.5r 




420 



4(0 



500 



Fig. 5. Absorption curves of: 

1. Thionin 

2. Methylene blue. 

3. Apparent mixture of these two dyes, incorrectly marketed (altho in good faith) 
as a dye intermediate between them in chemical composition. 

The absorption curves of typical yellow, orange, red, violet, and 
blue dyes are recorded in Figure 3. It will be noted that their maxi- 
mum absorption in each case falls within the range of the comple- 
mentary color (cf. Fig. 1). The great majority of dyes of these 

30 



colors, in the usual solvents and under the usual conditions, show 
but one absorption band in the visible spectrum. The curves are 
seldom perfectly symmetrical, however, and usually give indica- 
tions of localized secondary absorption in some portion of the band. 
It has been shown that this secondary absorption is due, in numer- 
ous instances, to a tautomeric form of the dve. It should never be 
accepted as evidence of the presence of a second dye unless it has 
been ascertained that it is not found with a pure sample of the dye 
under conditions of examination. 

The absorption curve of a green dye is recorded in Fig. 4. It has 
a principal band in the red and a secondary band in the violet. Both 
the absorption curve and the color of the dye could be matched 
closely by mixing a suitable blue and a yellow dye in the correct 
proportions. All green dyes absorb appreciable amounts of violet 
light as well as of red light. 

In Fig. 5 is given the absorption curve of a dye mixture, together 
with the curves of the component dyes. The mixture is reported to 
have been marketed in good faith as asymmetrical dimethyl 
thionin, a dye which is intermediate in constitution and in color 
between thionin and methylene blue (see methylene azure p. 48). 
The absorption curve plainly indicates the presence of two dyes, 
and suggests their pro})able identity. (It would be advisable to 
effect the separation of small amounts of both dyes, if their positive 
identification is desired.) The color of the mixture is very similar 
to that of dimethyl thionin. The absorption curve of that dye, 
however, is a simple and well defined curve resembling those of 
thionin and methylene blue, but occupying an intermediate posi- 
tion in the spectrum. 

This illustration shows how valuable the spectrophotometric 
analysis may be in determining whether a given product is a simple 
dye or a mixture of two or more dyes. This fact, together with its 
use in determining the exact shade of any dye, makes it the most 
valuable test to apply to a stain, other than to determine by actual 
use whether the sample will prove satisfactory to the micro.scopist 
or not. 



31 



CHAPTER IV 

DYES OF THE NITRO, AZO, AND OXYQUINONE GROUPS 

1. THE NITRO GROUP 

In this group the chromophore is -NO2. The chromophore is of 
such a strongly acid character that the dyes of this group are all 
acid dyes. The best known nitro dye is picric acid. 

PICRIC ACID c. I. NO. 7* 

Picric acid is formed by the action of nitric acid on phenol, 
thus introducing three nitro groups : 

OH 



O.N— r >— NO. 



NO. 

(An acid dye; absorption maximum about 360 in alcohol) 
This compound forms salts by the dissociation of the-OH group, 
and the salts have considerable value as stains. Ammonium 
picrate is the one most commonly thus used. 

Picric acid (or one of its salts) is quite extensively employed in 
contrast to acid fuchsin in the VanGieson connective tissue stain. 
It is also used as a general cytoplasmic stain in contrast to the 
basic dyes. It has further application as a fixative for tissues 
that are to be sectioned.! 

MARTIUS YELLOW C. I. NO. 9 

Synonyms: Manchester yellow, Naphthol yellow. 

OH 



—NO. 




NO. 

(An acid dye; absorption maxima about J^}^5, \39d, Sld\) 
Martins yellow has been used by Pianese in combination with 
malachite green and acid fuchsin for studying cancer tissue; the 
same technic was applied to plant tissue by Midler, and is now quite 

*This abbreviation stands for the number in the "Colour Index"; see Chapter 
II, p. 23. 

fFor bibliographic references concerning the procedures referred to in this 
chapter see Table 2 in Appendix I, pp. 110-128, and also the bibliography in 
Appendix III, p. 138. 

32 




I 

extensively used by plant pathologists in studying sections of 
tissue infected by fungi. The dye is also used in preparing certain 
light filters used in photomicrography. 

AURANTIA C. I. NO. 12 

Synonym: Imperial yellow. 

This dye is the ammonium salt of hexanitro-diphenylamine. 

NO, NO, 

\ 
O.N_/— \_N_ 

\_/ 

/ / 

NO, NO, 

(An acid dye; absorption maximum about J^25) 

It is obsolete as a textile dye and is almost unknown as a biological 
stain. It is called for, however, in combination with toluidine blue 
and acid fuchsin in the Champy-Kull technic for demonstrating 
certain cell constituents (mitochondria, etc.) 

2. THE AZO GROUP 

The azo dyes are characterized by the chromophore — X = X — 
joining benzene or naphthalene rings, thus: 

N = N 

\/\ 



It is possible for the azo group to occur more than once in a mole- 
cule, forming the disazo dyes, thus: 



N = N 





N = N 

The azo chromophore is distinctly basic; but not sufficiently so to 
make the dves basic when thev contain hvdroxvl radicals. Those 
containing amidogen radicals are, of course, pronouncedly basic. 
The position of the hydroxyl or amidogen group on a benzene 
ring in relation to the azo groHip is important. Ordinarily they are 
in the para position to each other, thus: 

N = N 





\/ \/\ 

OH 

The ortho position is next frequently assumed; rarely the meta 
position. When the hydroxyl group assumes the ortho position the 
character of the compound is quite distinct from that of the para 

33 



compounds. By a rearrangement of the atoms such a compound 
is sure to change to a quinoid form, thus : 

OH HO 

I I il 

_N = N_/\ /\_N=.N_/\ 



A compound of this latter structure cannot form salts and does not 
act as an ordinary dye. It does, however, prove to be soluble in 
oil and is able to color it by an apparently physical process. Hence 
the azo-ortho-phenols, or azo-beta-naphthols, like Sudan III and 
Sudan IV, 




N = N CH3 

N = N 




and 



H3C. 





are important fat staining dyes. 

ORANGE G. C. I. NO. 27 

Synonym: Wool orange 2G. 
Slightly different grade: Orange GG, GMP. 

S03Na 

/\_N = N 

I I ; 

HO" 

SOjXa 

(An acid dye; absorption maximum about Jf.85) 

This dye is strongly acid because of the two sulphonic groups. 
It is one of the most valuable plasma stains in histological work. 
It has great use as a background stain for haematoxylin and other 
nuclear dyes in cytology. It is frequently employed, both by 
botanists and zoologists, as a cytoplasmic stain, together with the 
two nuclear dyes safranin and gential violet in the Flemming triple 
stain. It is of importance to the pathologist for its use with anilin 
blue and acid fuchsin in the Mallory connective tissue stain; and 
is used in various other double and triple staining methods, such 
as that of Ehrlich-Biondi-Heidenhain, in which it is mixed with 
methyl green and acid fuchsin. The Erhlich "triacid mixture," 
also a combination of these same three dyes, is used in staining 
blood. A further use is Bensley's "neutral gentian," a combination 
of orange G and gential violet for staining the islands of Langer- 
hans. 

34 



BORDEAUX RED C. I. NO. S8 

Synonyms: Fast red B or P. Cerasin. Archelline 2B. 
Azo-hordeaux . Acid bordeaux. 

Various grades denoted: Bordeaux B, BL, G, R extra. 

N = N 
HO" 





NaSO,, SO,Na 

{An acid dye; absorption maximum about 520) 

Bordeaux red is used as a cytoplasmic stain, in particular when 
Heidenhain's haematoxylin is to be used immediately afterward 
as a nuclear stain. It has also been used by Graberg with thionin 
and methyl green for staining sections, particularly of spleen, 
testis, and liver. 

JANUS GREEN B. C. I. NO. 133 

Synonym: Diazin green. 

This is an azo dye having an azin as well as an azo chromophore 
group, and is thus related to the safranins. It is a compound of 
diethyl safranin with dimethyl anilin thru an azo group. 

CH3 
CH3 CH3 / 

Y^rVY rYY 

/\/ N \/\ /\/ CH3 

H.N / \ N = N 

CI 



(A basic dye; absorption maximum about 592.7) 

Janus green is best known for its use in demonstrating chondrio- 
somes, stained intra vitam, according to the technic of Michaelis, 
and as more recently developed by Cowdry and Bensley. It is 
also used by Faris with neutral red for sections of embryos. 

FAST YELLOW C. I. NO. l6 

{Echt Gelb) 

Synonym: Acid yellow. 
N = N SOjNa 




NaSOj NHa 

{An acid dye; absorption maximum about 4.90 in acid solution) 

35 



This dye is rarely used as a biological stain, but is called for by 
Schaffer for staining sections of bone, and by Unna in certain stain 
mixtures used in studying the phenomenon called by him chro- 
molysis. 

METHYL ORANGE C. I. NO. 1 42 

Synomyms: Orange III, Helianthin, Gold orange, Trapaeolin D. 

N = N CHj 

NaS03_| I I i_N 

\/ \/ \ 

CHj 

{A weakly acid dye; absorption maximum about 506 in 

acid solution) 

This dye has little use as a stain, but is widely employed as an 
indicator, as it is red in acid, and orange in alkaline solutions. Its 
chief value as an indicator is that it is sensitive to mineral acids 
without being affected by carbonates or most organic acids. It has 
been used by Bergonzini in the place of orange G in the Ehrlich- 
Biondi stain; and byEbbinghaus for staining keratin in sections of 
skin. 

ORANGE IV. C. I. NO. 1 43 

Synonyms: Orange N. Acid yellow D. Tropaeolin 00. 




NaS03 
{An acid dye; absorption maximum about 521 in acid solution) 

The onlv biological use of this dve seems to be occasionally as 
an indicator. 

ORANGE I. Ot^. no. 150 

Synonyms: Naphthol orange. Tropaeolin G. or 000 No. 1. 

N = N 



OH 

{An acid dye; absorption maximum about ^76) 

This is another dye which is turned red by excess of alkali and 
has therefore some use as an indicator. 

36 



ORANGE II. C. I. NO. 151 



Synonyms: Gold orange. Orange A, P, or R. Acid orange. 
Orange extra. Mandarin G. Tropaeolin 000 No. 2. 

N = N 

NaS03 \/\/ 

(^An acid dye; absorption maximum about Jlt.90) 

This dye, which differs from Orange I only in the position of the 
hydroxyl group on the naphthalene radical, is similar to it in color 
and properties, but does not change color with changing reaction 
of its solution. 



NARCEIN C. I. NO. 1 52 




{An acid dye) 

This dye is a derivative of Orange II, prepared from the latter 
by treatment with sodium bisulfite. It is rarely used either as a 
textile dye or in microscopic technic. It has been called for by 
Ehrlich, however, in combination with pyronin and methyl green 
or methylene blue to form a neutral dye. 

AMARANTH C. I. NO. 1 84 

Synonyms: Naphthol red. Fast red. Bordeaux. Bordeaux SF. 
Victoria rubin. Azo rubin. Wool red. 

N = N 
/HO \ 





NaS03 SOiNa 

NaSOj 

{An acid dye; absorption maximum about 525) 

Amaranth is not a commonly used stain, but is of considerable 
importance as a food color. It has been used by Griesbach for 
staining axis cylinders. 

37 





SUDAN III. C. I. NO. 248 

Synonyms: Sudan G. Tony red. Scarlet G or B. Fettponceau G. 

Oil red. Cerasin red. 

N=N_ 

I i HO 

N = N" 

{A iveakly acid dye; ahsorpiion maximum about 64^1, [590] ) 

In this dye the hydroxyl group is in the ortho position with 
respect to the azo group. As explained above (p. 34), such com- 
pounds show a tendency toward intramolecular rearrangement so 
that the hydrogen atom detaches itself from the hydroxyl group 
and becomes fixed to the neighboring nitrogen. Such a compound 
is neither acid nor basic, and not being able to form salts is not an 
ordinary dye, but is fat soluble and has the power of coloring fat. 
This fact gives Sudan III its chief value to the histologist. It was 
introduced as a fat stain by Daddi in 1896. 

For some time Sudan III was the only important fat stain 
known. More is now known in regard to fat soluble stains, thanks 
to the research of Michaelis (1901). It was he who showed the 
relation of this property of certain dyes to their lack of basic or 
acid character. He showed that new dyes with this property and 
of greater staining power might be built up synthetically by taking 
advantage of the fact that the azo group will attach itself in the 
ortho position if the para position is already occupied. In this 
way azo-ortho-phenols and beta-naphthols can be prepared, and 
they prove to be fat soluble. Michaelis suggested the following 
dye, which has now to a considerable extent replaced Sudan III. 

SUDAN IV c. I. NO. 258 

Synonyms: Scarlet red. Scharlach R. Oil red IV. 

Fettponceau. Ponceau SB. 

CH3 

I N = N 




H.C 



(A weakly acid dye; absorption maximum about 657. 4, [605.5] 

in H^SOa) 

This di-azo naphthalene compound is similar to Sudan III ex- 
cept that it is a dimethyl derivative. This fact makes it a deeper, 
more intense stain; but having the hydroxyl group in the ortho 

38 




position, it has similar physical properties and is fat soluble. It is, 
therefore, one of the best fat stains knoTVTi. 

BIEBRICH SCARLET, WATER SOLUBLE C. I. NO. 280 

Synonyms: Croceine .scarlet. Scarlet B. or EC. Ponceau B. 

Double scarlet. 

X = X 

" r{ X 

/\/ I I H0_ 

xXaS03 /\/ 

XaSOj N = N 

{An acid dye; absorption maximum about 503.5) 

The chief biological application of this dye is for medicinal pur- 
poses, but it is occasionally used as a plasma stain, notably for 
tissues after staining with polychrome methylene blue or Unna's 
haematein. It has also been made use of bv Paladino mixed with 
alum haematoxylin for double staining effect on histological, 
material. 



BISMARCK BROWN Y C. I. NO. 33 I 

Synonyms: Ve.suvin. Phenylene brown. Manchester brown. 
Excelsior brown. Leather brown. 

Slightly different shade: Bi-wiarck broivn G. 

To be distinguished from :Bismark brown R or GOOO (C. I. No. 
332.) 

{An acid dye.) 

The various shades of Bismarck bro\\Ti are mixtures of different 
compounds, the most important of which are salts of the following: 

XH2 




This dye was formerly employed quite extensively as a contrast 
stain, but has now been replaced to some extent by others. It is 
still used, however, as a mucin stain, and is good for vital staining 
and for staining in bulk. It is employed in staining cellulose walls 
of plants in contrast to haematoxylin; and occasionally for staining 
bacteria in contrast to gential violet in the Gram technic. 



39 



CONGO RED 



C. I. NO. 370 



Synonyms : Congo. Cotton red. Direct red. 

NHa 



/ 



N = N 





X 



\. 



I NaS03\/\/ 



NaS03_ 





\ 
/ 



\ /\ 

N = N 

NH. 

{An acid dye; absorption maximum about 4-85.) 

This dye is best known to the biologist as an indicator. The 
dye acid is blue, but its sodium salt is red. The red color of the 
salt is readily changed by weak acids into blue. Besides serving 
as an indicator, congo red has certain histological uses, as for axis 
cylinders (Griesbach) for embryo sections (Schaffer), for staining 
plant mucin, and as a general background stain in contrast to 
haematoxylin and other nuclear dyes. It has been used by Klebs 
as a reagent for cellulose. 



TRYPAN RED 



C. I. NO. 438 



S03Na 




NaSOj SO^Na 

NaSOi 
(An acid dye.) 

The chief use of this dye is as a vitstl stain. 



SOiNa 



C. I. NO. 448 • 



BENZOPURPIN 4b 

Synonyms: Cotton red J^B. Dianil red 4-C. Diamin red 4^. 

Sultan 4^B. Direct red I^B. 

NH, NH. 

I N = N_/~\_/~\_N = N I 



CH3 



CH3 



NaS03 NaS03 

{An acid dye; absorption maximum about 497.) 



40 




This dye has also been used for vital staining; and has been 
employed by Zschokke as a plasma stain especially in contrast to 
haematoxylin. 

TRYPAN BLUE C. I. NO. 477 

Synonyms: Chlorazol blue SB. Benzo blue 3B. Dianil blue H3G. 

Congo blue 3B. Naphthamine blue 3BX. Benzamine 

blue SB. Azidine blue SB. Niagara blue SB. 

HaN OH HO NHa 

\ I N = N_/~V_/-"\_N = N I / 

CH3 CHj /\/\/\ 

NaSOi SOiNa NaSO^ SOjNa 

{An acid dye.) 

Apparently the only biological use of this dye is in vital staining. 

Other azo dyes sometimes mentioned in connection with hist- 
ology are: 

Janus red; C. I. No. <im 

Tropaeolin O; C. I. No. 148. Syn: Chrysoin. Gold yellow. Acid 
yellow 

Tropaeolin Y; C. I. No. 148 (see note). 

Roccellin; C. I. No. 176. Syn: Fast red A, AV, or 0. Cerasin, 
Rubidin. Cardinal red. 

Crystal ponceau 6R; C. I. No. 89; Syn: Ponceau 6R. 

Carmin naphtha; C. I. No. 24. Syn: Sudan 8. Scharlach B. 
Oil yellow. 

Alizarin yellow GG; C.I. No. 36. Syn : Anthracene yellow. Ben- 
zene yellow. 

Chrysoidin R; C. I. No. 21. Syn : Cotton orange. Cerotin orange. 

Chrysoidin Y; C. I. No, 20. Syn: Brown salt R. Dark brown 
salt R. 

Alizarin yellow R; C. I. No. 40. Alizarin orange. Benzene yel- 
low PN. Orange R; Anthracene yelloiv RN. 

Diamond flavine; C. I. No. 110. 

Diamond black F; C. I. No. 299. Syn.: Salicin black. Chrome 
black. 

Niagara blue 4B; C. I. No. 520. Syn. : Niagara sky blue. Benzoin 
sky blue. Dianil blue H 6 G. Congo sky blue. Naphthamine blue. 

41 



3. THE OXYQUINONE GROUP 
The oxyquinone dyes include derivatives of anthracene, 




thru its oxidation product anthraquinone 

O 



O 

These dyes are the first to be considered here in which the quinoid 
structure occurs. The quinoid ring, which is the most important 
chromophore in nearly all the dyes to be discussed in the three fol- 
lowing chapters, forms very strong chromogens, which require 
only the addition of auxochrome groups to be converted into strong 
dyes, either basic or acid. The chromogen anthraquinone is con- 
verted into a dye by the addition of hydroxyl groups, its best 
known derivatives among the dyes being: 1:2 dihydroxy-anthra- 
quinone (alizarin) and 1 :2 :4 trihydroxy-anthraquinone (purpurin). 
Both of these compounds occur in nature in the root of madder, 
being the colored principles of madder extract. They have the prop- 
erty of combining with metalic oxides to form so-called "lakes", 
insoluble compounds of different color from the dye entering into 
them. This makes them valuable ones to use after mordanting 
with aluminium, iron or chromium compounds. 



ALIZARIN 

O OH 

II / OH 



C. I. NO. 1027 



II, 

O 

{An acid dye; absorption maxima about [610.S], 566.5, [5S7.6] 

in alkaline solution.) 

Alizarin stains tissues a feeble yellowish red if used on them 
directly. In the presence of aluminium compounds intense red 
colors are formed; bluish violet in the presence of iron; and brown- 
ish violet in the presence of chromium. It has been used as a stain 
for nervous tissue. The chief present use of alizarin, however, is 
as an indicator. 

42 



ALIZARIN RED S 



Synonyms: Alizarin red, water soluble. 

Alizarin sulphate. 



c. I. NO. 1034 
Alizarin carmin. 




SOjNa 



{An acid dye.) 

This dye, sodium alizarin sulphonate, is used by Benda for stain- 
ing chromatin in combination with crystal violet, the chromatin 
staining brown, while the mitochondria stain violet. It is also 
used as a vital stain for nervous tissue in small invertebrates, and 
by Schrotter for sections of nervous tissue. 

PURPURIN c. I. NO. 1037 

Synonyms: Alizarin No. 6. Alizarinpurpurin. 




(An acid dye; absorption maxima about [521.1], Jf85.5, 

[Jf.o5.o\ in alcohol.) 

Purpurin is very similar to alizarin, but forms scarlet red lakes 
with alumina. It has been used as a nuclear stain for histological 
material, and for determining the presence of insoluble calcium 
salts in the cell contents. 




43 



T 



CHAPTER V 

THE QUINONE-IMIDE DYES 

HE dyes of the quinone-imide group contain two chromophore 
groups, the indamin group — N = , and the quinoid benzene 
ring 




They are derivatives of the theoretical compound paraquinone 
imide, which, if it existed in its free state, would have the formula 

HN_/"~V=NH 

In the typical indamin formula one of the imide hydrogen atoms is 
replaced by a phenyl group, thus: 

/^ N_/— \_NH 





In the thiazins the introduction of a sulfur atom, attached to 
both the phenyl and the quinone groups, forms a third closed ring, 



as: 



/\_S _/\=NH 



■N= 



imido-thio-diphenylimide 

In the oxazins, an oxygen atom takes the place of the sulfur of the 
thiazins, thus: 

\/\_0_/\=NH 




oxazin 



1. THE INDAMINS 

No dye in this group is a common biological stain. The follow- 
ing are occasionally mentioned, however, in connection with 
histology : 

Bindschedler's green. A tetramethyl indamin. C. I. No. 819. 

Toluylene blue. A diamido, dimethyl indamin. C. I. No. 820. 

2. THE THIAZINS 

The thiazins constitute one of the most important groups of 
dyes from the standpoint of the biologist; while for textile dying 
the group contains but a small number of dyes of any importance. 
In these compounds, as mentioned above, the two benzene rings 
are further joined by a sulfur atom. 

44 



THIONIN C. I. NO. 920 

Synonym: Lauth's violet. 
(A basic dye; absorption maximum about 602.)* 

Thionin, having two amino groups, is a strongly basic dye. The 
exact structural formulae of this dye and its derivatives, as well as 
many others in which two benzene rings are similarly joined, are in 
some dispute. At least two types of formulae are possible for the 
thiazins and oxazins, as well as for the xanthene dyes (Chapter 
VII). One t>pe is known as the orthoquinoid, the other as the 
paraquinoid. 

It will be recalled (see p. 13) that when the quinoid ring is formed 
the two hydrogen atoms replaced by atoms or groups with double 
valency bonds may be either in the para or in the ortho position to 
each other. It will also be recalled from elementary chemistry 
that sulfur and oxygen may be either bivalent or tetravalent. These 
facts make it possible for a thiazin or an oxazin to have either one 
or the other of the different structures represented by the following 
two formulae for the theoretical thionin base: 



OH 




paraquinoid formula 
OH 



H.X 



NH. 



"N= 



orthoquinoid formula 
In the case of the paraquinoid formula the compound is an am- 
monium base of the type discussed on p. 15, which is capable of 
salt formation thru its pentavalent nitrogen. In the case of the 
orthoquinoid formula the salt formation takes place thru the 
tetravalent sulfur, the base being of the type known as a sulfonium 
base. There are arguments in favor of either formula, and from 
the standpoint of the biologist it does not matter which is preferred. 
Possibly both forms actually exist simultaneously. For the sake of 
uniformity the paraquinoid form will be sho^-n in the following 
pages wherever possible; but with the understanding that the 
orthoquinoid form is equally permissable. 

The dye, thionin, is a salt, generally a chloride, of the above 
mentioned base; and on the assumption of paraquinoid structure, 
it has the following formula : 

H.X /\ S _/\=XH, 

I 
CI 



"See Fig. 5. p. 30. 




It is no longer used as a textile dye, and is very carefully to be 
distinguished from thionin blue (C. I. No. 926) which is known to 
the trade and is sometimes furnished in place of the desired dye 
when thionin is ordered. Thionin is an especially valuable dye for 
histological work on account of its metachromatic properties, that 
is its ability to impart different colors to different histological or 
cytological structures. It is a very valuable chromatin and mucin 
stain, proving especially useful in staining the tissue of insects; and 
is recommended by Ehrlich because it stains amyloid blue but mast 
cells and mucin red. It is a useful vital stain. Perhaps its greatest 
value at the present time is in the staining of frozen sections of 
fresh animal or human tissue, particularly in the study of tumors. 
It is also used by Frost for staining very young bacterial colonies 
in his "little plate" technic for coointing bacteria. (Unfortunately 
Frost specifies thionin blue in one of his papers, altho the latter 
proves entirely unsatisfactory for the purpose.)* 

METHYLENE BLUE C. I. NO. 922 

Synonym: Swiss blue. 

Various grades denoted: Methylene blue BX, B, BG, BB; grade 
preferred for biological work: Methylene blue Med. U. S. P. 

Methylene blue is a salt of tetramethyl thionin (generally a 
chloride, altho other salts are known, such as sulfates). On the 
assumption of the paraquinoid structure, it has the formula: 




{A basic dye; absorption maximum about 665.) 

According to the general rule as to the influence of methylation on 
color it is less red in shade than thionin and is therefore a purer 
blue. Its absorption curve has a maximum at about 665^tju, with 
a lesser peak at about GlO^^t. The methylene blue of commerce is 
generally a double salt, the chloride of zinc and methylene blue. 
The zinc is toxic, however; so for some time the zinc-free methylene 
blue chloride has been prescribed for medicinal purposes; hence the 
meaning of the term Methylene blue Med. U. S. P. The zinc 
double salt is less soluble, particularly in alcohol, so for most stain- 
ing purposes is less desirable. The investigations of the Commis- 
sion show that for all ordinary staining purposes the zinc-free 
compound is best; so that is the form at present recommended. 

*For bibliographic references concerning the procedures referred to in this chap" 
ter, see Table 2 in Appendix I, pp. 110-128, and also the bibliography in Appendix 
III, p. 138. 

46 



Methylene blue is perhaps the stain which the pathologist and 
bacteriologist would have the greatest difficulty in doing without, 
and it is of great value to the zoologist as well. It is employed for 
a greater variety of purposes than any other biological stain except 
possibly haematoxylin; and for this reason was the first dye to be 
given a thoro investigation by the Commission. It is used: first, 
as a nuclear stain in histology, for which purpose its strongly basic 
character as well as the ease with which it can be applied without 
over-staining, make it quite valuable; secondly, as a bacterial 
stain, notably in milk work and in the diagnosis of diphtheria, 
where it is especially useful because it has an affinity for the bac- 
terial protoplasm as great as that of the rosanilin dyes, but is less 
intense, more selective in its action and more subject to differen- 
tiation; thirdly in the vital staining of nervous tissue, where a non- 
toxic, basic dye is needed; fourth, in combination with eosin in the 
blood stains, thanks to the ease with which it can be partly con- 
verted into other dves like methvlene violet and methvlene azure, 
and thus acquire polychrome properties; and lastly as an indicator 
in the Levine eosin-methylene-blue medium for differentiating the 
colon and aerogenes organisms. 

The polychrome properties just mentioned are quite likely to 
develop in a methylene blue solution upon standing. Anyone who 
has had much experience with the stain is familiar with the oc- 
casional green tones from methylene green, the reddish shades of 
methylene azure (azure I) and methylene violet. Such a solution 
is known as "polychrome methylene blue." Its formation is 
hastened by boiling with alkali. In preparing blood stains the 
methylene blue solution is treated for this purpose with sodium 
carbonate, and then eosin is added, which enters into chemical 
combination with the other dyes present, inasmuch as eosin is an 
acid dye while methylene blue and its derivatives are basic. The 
combination of eosin and methylene blue is often spoken of as the 
eosinate of methylene blue. (For a more detailed discussion of the 
subject see Chapter VIII.) 

It can be readily understood that an especially pure product is 
needed when the dye is to be used for vital staining or in blood 
work. For vital staining the U. S. P. zinc-free dye is always rec- 
ommended, sometimes with even further purification; altho the 
recent investigations carried on by the Commission indicate that 
the U. S. P. product is sufficiently pure. For blood work there is 
frequently recommended a "methylene blue rectified for blood 
stains." This grade, however, is generally less pure than the 
medicinal or U. S. P. grade, and there seems no reason for specify- 
ing it. The same is true of various other grades such as those 
denoted BX, BG, etc., which are ordinarily purer than the textile 
dye, but less pure than the medicinal grade. 

In a recent paper by Scott and French (IQ'^-tb) it is claimed that 
the specially desired staining properties of methylene blue are 

47 



associated with the presence of lower homologs, particularly the 
dimethyl thionins. One of the dimethyl thionins is methylene 
azure A; hence the statement of Scott and French is merely another 
way of saying that methylene blue should be partially polychromed 
in order to have its best staining powers. These lower homologs 
are generally present to some extent in methylene blue as sug- 
gested by the minor peak in the absorption curve at 610jLl^t. Scott 
and French show that a methylene blue may be specially prepared 
which contains more than usual of these lower homologs and that 
it is better than the ordinary product for their purposes. How 
widely their results can be applied to all microscopic uses of methy- 
lene blue cannot be decided at present. It is interesting that one 
particular instance has come to the attention of the Commission in 
which a pure methylene blue was unsatisfactory for a certain 
neurological procedure, but a crude textile methylene blue proved 
satisfactory. This may possibly have been due to the presence of 
other dyes in the impure sample. 

One serious bit of confusion has arisen from the designation 
^'methylene blue for bacilli" which was used on a certain type of 
methylene blue imported before the war. This was the label 
placed on a certain type of zinc salt, containing a small amount of 
free chloride. Its designation seemed to imply that it was especially 
adapted for staining bacteria; recent investigations indicate that 
it should rather be considered not good enough for any other pur- 
pose! Even for staining bacteria it is not especially satisfactory; 
for the most common methylene blue solution of the bacteriologist 
is the Loeffler formula, in which a certain amount of saturated al- 
coholic solution is used as a stock. Now, since the zinc salt is 
nearly insoluble in alcohol, such a stock solution contains little but 
the free methylene blue chloride present. For all these reasons the 
discontinuing of this grade of methylene blue is decidedly to be 
recommended. 



METHYLENE AZURE* 



Synonyms: Azure I. Azure A. Azure B. 

This is one of the components of polychrome methylene blue, 
first described by Bernthsen (1885). Definite knowledge of its 
chemical nature was gained by Kehrmann (1906) and Bernthsen 
(1906). The former showed that there are two azures, the asym- 
metric dimethyl thionin. Azure A, and trimethyl thionin, Azure B. 
The symmetrical dimethyl thionin, which he prepared, was found 
to belong in a quite different category. 



*For the following statements in regard to methylene azure and methylene violet 
the author is indebted to Dr. W. J. MacNeal. 

48 



CH, 



\ 



N 



CH, 



.S. 






CH3 










CH3 


\ 




H 


/ 


N 




/ 


N H 


/ \/\ s 


/\ 


N H 




CH3 










CI 










CI 



Tri-methyl thionin 
(Azure B, Kehrmann) 



Asymmetric di-methyl-thionin 
(Azure A, Kehrmann) 



CH, 



H 



\ 

I 

/ 



N 



X^ 




Symmetrical di-methyl thionin 
(not an azure dye) 

Bernthsen (1906) and MacNeal (1906) simultaneously and in- 
dependently described the easy method of preparing azure by 
oxidizing methylene blue by chromate in acid solution and Mac- 
Xeal (1925) has perfected this method so that it is now possible to 
obtain large yields of Azure A and a fair yield of Azure B. 

Azure I (Giemsa) is a trade name applied to a secret prepara- 
tion which appears to be a somewhat variable mixture of Azure A 
and Azure B. Azure II (Giemsa) is an intentional mixture of 
Azure I (Giemsa) with an equal quantity of methylene blue. 

The azures are important constituents of all the polychrome 
methylene blue stains and are present in undetermined and variable 
amounts when these solutions are empirically prepared. For the 
preparation of the tetrachrome blood stain of MacXeal (see p. 90), 
a definite quantity of Azure A (Asymmetric di-methyl-thionin) is 
required. 

METHYLENE VIOLET (Bcrnthscn) 

Methvlene violet is formed whenever methvlene blue is heated 

with a fixed alkali or alkali carbonate. It is a feeble base with the 

formula 

CH3 



N 



CH, 




.0 



"N= 



Its preparation from methylene blue is more difficult than that of 
Azure A. A fair yield (30 to 40 per cent) may be obtained by 
oxidizing methylene blue in dilute ammoniacal solution with 
potassium chromate and then driving off the ammonia by boiling 
with the addition of sodium carbonate. It may also be prepared 
from Azure A by boiling this with dilute alkali carbonate. Methy- 
lene violet precipitates out as needle crystals, insoluble in water. It 



49 



may be recrystallized from ethylene dichloride (C2H4CI2) in which 
it forms a deep carmine red solution. Although insoluble in water 
when pure, methylene violet is soluble when mixed with methylene 
blue or with the azures. It plays an important part in the nuclear 
and granule staining of the polychrome methylene blue stains. A 
definite quantity of this dye is employed in the tetrachrome blood 
stain of MacNeal. 

Methylene violet (Bernthsen 1885) is not a textile dye and must 
not be confused with methylene violet RRA or 3RA, which is C. I. 
No. 842. 



CH3 



CH, 



METHYLENE GREEN 
NO. 



C. I. NO. 924 



CH3 



N_/\_S _/\ = N-CH, 



CI 



-N=\/ 
{A basic dye; absorption maxima at about 660, 607.) 

This dye is a mono-nitro methylene blue, obtained by the action 
of nitrous acid on methylene blue. The formula is probably as 
given above, but the exact position of the nitro group is uncertain. 

It is occasionally used as a substitute for methyl green, especially 
by botanists in the case of wood and fixed chromatin, and gives 
good results in combination with eosin. 

TOLUIDINE BLUE O C. I. NO. 925 

Synonym: Methylene blue 0. 

This dye is closely related to thionin and to methylene blue in 
structure, and even more closely to methylene azure A : 

CH3 

\ 



N 



CH, 



"N= 



,NHaCl 
CH. 



{A basic dye; absorption maximum about 635.) 

Toluidine blue is not ordinarily used for textile dyeing, but is more 
easily prepared than thionin — a fact of considerable importance, 
as it has properties very much like the latter. It proves, in fact, 
that it can be substituted in many ways for thionin, as for example 
in staining frozen sections of fresh tissue. It is quite a useful stain, 
being an important ingredient of Pappenheim's panchrome stain 
for tissues and blood, and also the main constituent of the Albert 
stain, which is at present replacing methylene blue in the diagnosis 
of diphtheria. 



50 



NEW METHYLENE BLUE N 

Synonym: Methylene blue NN. 



c. I. NO. 927 



CHi'CHa 



\ 



H 



CH3 CH3 CH.CH3 

. I I / 

N /\_S_/\=N_H 

CI 



(A basic dye; absorption maxima about [6S6.4], 588.) 

This dye has practically never been called for in microscopical 
work. The most interesting fact concerning it which has come to 
light relates to the VanWijhe technic as applied by Louise Smith 
(1920) for staining the cartilage of frogs. The latter specified 
methylene blue, but the results could not be duplicated with any 
domestic or foreign methylene blue subsequently obtained. When 
furnished thru the Commission with samples of various stains to 
try, it was found that her earlier results could be duplicated with 
new methylene blue — a fact which not only implies mislabeling of 
her original supply of methylene blue, but suggests that new methy- 
lene blue may have some value in histological work. 

3. THE OXAZINS 

This group is like the thiazins in chemical formula except that 
the sulfur atom is replaced by an oxygen atom. Only a few of the 
dyes find use in microscopic technic, and they are not stains having 
very general application. 

BRILLIANT CRESYL BLUE C. I. NO. 877 

Synonyms : Cresyl blue 2RN or BBS; brilliant blue C. 

CHj'CHa 



N 



CH.CH 



O 



.NH, 



3 ^AA2 



CH. 



CI 



(.4 basic dye; absorption maxima about 631.8 [579.5]) 

This dye is prized for certain special work on account of its 
highly metachromatic properties. Its chief biological use is for 
staining blood to bring out the platelets and the reticulated blood 
corpuscles. 

Brilliant cresyl blue proved one of the most difficult stains to 
obtain in good quality since the war. The problem was finally 
solved however, and the pre-war stain has not only been equalled 
but surpassed. 



51 



NILE BLUE SULFATE C. I. NO. 913 

Synonym: Nile blue A. 




{A basic dye; absorption maxima about 64-^.5, [592.2] ) 

The use for which this dye is best kno^^'n to the biologist is the 
Lorrain Smith fat stain. In this procedure the dye is boiled with 
dilute sulfuric acid, and thus hydrolyzed, with the introduction of 
oxygen in the place of the radical XHo (S04)k> in other words pro- 
ducing a new dye of the class known as oxazones. This oxazone 
dye is red, and is fat-soluble. Nile blue sulfate itself, on the other 
hand, is not fat-soluble but combines readily with fatty acids. As 
a result the technic serves to distinguish between the free fatty 
acids in histological material and the neutralized fats, the former 
staining blue, the latter red. 

Nile blue sulfate is also used unaltered for staining living tad- 
poles previous to making transplants, in order to distinguish the 
grafts. 

CRESYL VIOLET 

Synonym: Cresylecht violet (i.e., cresyl fast violet). 
{A basic dye; absorption maximum about 585.) 

No information is at hand concerning the exact chemical form- 
ula of this dye. It is understood to be a derivative of brilliant 
cresvl blue. 

Cresyl violet is not a widely used stain, but finds some employ- 
ment on account of its strongly metachromatic properties. It is 
valuable in making permanent preparations of nervous tissue. 
According to Ehrlich (1910, II, p. 78) it stains nuclei violet, plasma 
blue, amyloid, mucin and mast cell granules red. Williams (19*23) 
uses it for staining sections of fresh tumor tissue. 

As cresyl violet is not a textile dye, some difficulty has been 
found in obtaining it for biological purposes. Williams reports con- 
siderable trouble in this respect. Spectrophotometric examination 
of the pre-war material used by Williams shows it to have been a 
mixture, apparently of cresyl violet with another dye of more red- 
dish cast. (See Ambler and Holmes 1924.) The domestic sample 
with which he obtained unsatisfactory results on account of its 
entire lack of metachromatic properties was this unknown red dye 
alone; while the domestic sample with which he obtained good 
results, altho not identical with those obtained with the pre-war 

52 



stain, was true cresyl violet without the reddish dye. True cresyl 
violet, like this last mentioned product can now be obtained in 
constant quality in America, and proves to have all the properties 
needed in this stain. 

Other oxazin dyes sometimes mentioned in connection with 
histology are: 

Capri Blue. C. I. No. 876. 

Naphthol Blue. C. I. No. 909. Synonym : Neiv blue B, Fast blue 
SR. Phenylene blue. Meldolas blue. Indin blue 2RD. 

4. THE AZIXS 
The dyes of the azin group are derivatives of phenazin, 
C6H4N2Cf,H4, a compound containing two benzene rings linked 
thru two nitrogen atoms in such a way as to form a third ring. 
Two formula are possible: 



N_ 



"N 



and 




In the case of the first formula the quinoid ring is the chromo- 

_N— 

phore; in the case of the second formula the azin group itself, | , 

(see p. 17) is assumed to be the chromophore. The quinoid formula 
is generally preferred today. 

Phenazin is weakly basic, but is not a dye as it does not contain 
auxochrome groups. In other words, it is a chromogen. Either 
an -OH group or one or more -NHo groups may be introduced to 
give it dye properties. The acids and bases are very weak if there 
is only one auxochrome group present, and their salts are readily 
decomposed. For this reason some of them are of use as indicators. 
Strong bases are encountered only among the safranins where basic 
character is derived not only from the two -NH2 groups but also 
from one of the azin nitrogen atoms which becomes pentavalent 
and takes part in salt formation. 



a. 



Amido-azixs or Eurhodins 

If one or more amino groups are introduced into a phenazin, a 
dye is formed of the class known as eurhodins. They are very weak 
bases, and therefore weak dyes; but as their salts are readily de- 
composed with a resulting color change, they form useful indicators. 
The best known of the group is toluylene red, base : 



CH, 



CH3 



\ 
/ 



N 



NH2 
_CH3 



53 



The chloride of toluylene red is the well known neutral red. 



NEUTRAL RED 



C. I. NO. 825 



CH3 



CH, 



N 




{A weakly basic dye.) 



CH, 



The name of this dye comes from its characteristic neutral color 
which is neither red or yellow. It is yellow in solutions a little 
below the neutral point (i.e., pH=7.0) in reaction and red in weak 
acids, even the reaction of ordinary tap water being sufficient to 
bring out the acid color; at a higher range of acid it turns blue. 
This gives it some value as an indicator. As an indicator it is also 
used in bacteriological media for distinguishing the colon from the 
typhoid organisms, and for recognizing other forms; altho it is 
employed for this purpose much less today now that other dyes 
have been shown to have even greater value for the same type of 
work. 

As a stain it has special value where a weakly basic, non-toxic 
dye is called for. It is used as a vital nuclear stain; for the "vital'* 
staining of blood, that is of fresh blood observed under a micro- 
scope in a moist chamber; and for staining fresh gonorrhoeal pus 
under similar conditions. It is used for bringing out the Nissl 
granules in nerve cells; it also has some use in general histological 
staining, especially for embriological tissue in combination with 
Janus green, as recommended by Faris. 



NEUTRAL VIOLET 



CH, 



\ 



N_/\_N=/\_NH. -HCl 



CH, 




N 



-NH— 



"N^ 



C. I. NO. 826 
CH3 



CH3 



(A weakly basic dye.) 

This dye is very similar in its properties to neutral red, except 
that, due to its greater molecular weight, it is more bluish, giving 
a violet instead of a red color. It can be used as an indicator, but 
has been seldom used in histology. Unna (1921) however, has 
recently used it in a dye mixture employed in the study of chro- 
molysis. 

b. Safranins 

Quite a long series of azin dyes are known in which one of the 
nitrogen atoms of the azin group is pentavalent and another ben- 



54 



zene ring is attached to it. This pentavalent nitrogen allows the 
compounds to behave like ammonium bases; so with the amino 
groups which are always present, the basic properties of these dyes 
are very strong. The theoretical base of the simplest safranin 
would have the formula : 

N=/\. 




H.N / \ NH, 

OH 



This form of ammonium base does not actually exist, as the safranin 
bases really occur in the form of anhydrides; but salts of these 
ammonium bases are the commonly known dyes. The commercial 
dyes are ordinarily chlorides. 

There are two groups of safranins : the benzo-saf ranins in which 
the azin group unites two benzene rings; and the naphtho-saf ranins 
in which it unites two naphthalene groups. The simplest safranin 
is pheno-safranin, which is the chloride of the theoretical base just 
given, namely: 

N_ 



H,N / \ NH 




The commercial safranins are ordinarily methyl or ethyl substitu- 
tion products of this; or occasionally phenyl substitution products. 
The one of greatest value to the biologist is generally called Safra- 
nin O. 

SAFRANIN O C. I. NO. 84 1 

Slightly different shades: Safranin AG, T, MP, Y, and G. (Altho 
all included in C. I. No. 841 they are different from the grade 
here described.) 

(A basic dye; absorption maximum about 515.) 

The common safranins of commerce, under various shade 
designations, are mixtures of di-methyl and tri-methyl pheno- 
safranin : 

CH3 






"N=\/\ 
H,N / \ NH3 H^ / \ NHa 

/\ CI 



55 



The shade differs according to the proportion of these compounds 
present, the red being deeper according to the proportion of the 
tri-methyl compound in the mixture. The type safranin O, which 
proves best for ordinary biological purposes, can be defined as 
having its absorption maximum at 515///i. 

Safranin O is one of the most important nuclear stains knowTi to 
the histologist. The botanist finds it especially valuable, as it 
brings out lignified and cutinized tissues in vascular plants, and 
can be employed in combination with a variety of contrast stains; 
it is valuable as a protein stain in plants, and can be used to stain 
spore coats. The cytologist makes use of it in the Benda technic 
to stain chromatin in combination with light green as a contrast 
stain; and even more widely in the Flemming triple stain, in which 
it is employed as a chromatin stain, together with gentian violet 
and orange G. The bacteriologist has some use for it, especially 
as a counterstain in the Gram technic (see p. 68). 

AMETHYST VIOLET C. I. NO. 847 

Synonyms : Heliotrope B, Iris violet. 
This dye is tetra-ethyl pheno-saf ranin : 

/\ N_/\ 

CH3CH. [ I II CH.CH3 

\ /\/~N=-\/\ / 

N / \ N 

/ /\ CI \ 

CH.CH, I I CH3CH2 

{A basic dye; absorption maxima about 589, [5Ji.5.5] ) 

Amethyst violet has been used by Ehrlich and Lazarus as a basic 
dye in certain triple staining technics. 

A further dye of this group which the biologist must take into 
account, altho it seems to have no significance as a stain, is methy- 
lene violet RRA or 3RA, C. I. No. 842 {sy n. : fuchsin or safranin 
extra blue.) This dye is a di-methyl safranin in which the methyl 
groups are introduced into one of the amino groups instead of 
directly into the benzene ring. It has no connection with the 
methylene violet of Bernthsen, which is one of the constituents of 
polychrome methylene blue; see p. 49. 

MAGDALA RED C. I. NO. 857 

Synonyms: Naphthaline red, naphthaline pink, naphthylamine 

pink, Sudan red. 

This is a naphtho-saf ranin, and is a mixture of the monamino 
and diamino compounds: 

56 





XHa H.X_ I I If NH. 

and 



(A basic dye; absorption maximum about 521^.) 

A true Magdala red put on the market before the war under the 
name of Magdala red echt is very expensive. According to Cham- 
berlain (19'24 page 58) this is less satisfactory in botanical work 
than a cheaper form of Magdala red formerly available not labeled 
"echt." Chamberlain further states that he has been able to 
obtain recently results with phloxine identical with those which he 
used to be able to obtain with the less expensive form of Magdala 
red. A sample of the latter examined by the Commission proves 
apparently to be erythrosin, — in other words an acid dye of an 
entirely different group and very closely related to phloxine. This 
makes Dr. Chamberlain's failure to obtain results with Magdala 
red echt entirely comprehensible. (See also discussion under 
phloxine and erythrosin page 81-8'2.) 

Magdala red is used by botanists with anilin blue, in staining 
algae. It was used by Flemming as a nuclear stain, and by Kult- 
schitzky for staining elastic tissue. 

c. THE IXDULIXS 

Indulins are similar to safranins but are more complex: being 
quite highly phenylated amino derivatives. The only one to 
concern us is: 

XIGROSIX, WATER SOLUBLE C. I. NO. 865 

Synonyms: Xigrosin W, WL, etc. Gray R, B, BB. Silver gray. 

Steel gray. Induliri black. 

{A basic dye; absorption maximum about 587.) 

The exact constitution of nigrosin is uncertain. It is recom- 
mended by Ehrlich for staining the tissue of the central nervous 
system either alone or in combination with other stains, and by 
Jarotsky for staining pancreatic tissue following haematoxylin . 
Botanists use it in studying algae and fungi. Pfitzer's picro- 
nigrosin serves as a chromatin stain. Nigrosin is also used by 
Unna in combination with ''orange" (orange G?) in the study of 
the process of chromolysis. 



57 



CHAPTER VI 

THE PHENYL METHANE DYES 

ONE of the most important groups of dyes, both from the stand- 
point of the dyer and from that of the biologist, is a group of 
substituted methanes, or in other words compounds with a 
central carbon atom. In methane, CH4, it is possible to replace 
any of the hydrogen atoms with methyl, ethyl, or phenyl groups. 
If one H is replaced with CH3, it becomes ethane, CHs-CHs- 
If two are replaced with CH3 groups it becomes propane, 
'CH3CH2CH3; while if there are three substituent CH3 groups 
it becomes iso-butane: 

CH, CH, 

\ / 
C 

/ \ 
CH3 H 

Similarly if one H is replaced with a phenyl group it becomes 
phenyl methane or toluene: 



_CH3 




if with two it becomes di-phenyl methane 




if with three it becomes tri-phenyl methane; 




Certain substitution products of the di- and tri-phenyl methanes 
are among the most powerful dyes known. 

Di- and tri-phenyl methane, themselves, are not dyes, nor are 
they chromogens. They lack both the chromophore and the auxo- 
chrome groups. The first step (theoretically) in converting them 
into dyes is to introduce an -OH group in the place of one of the 
unsubstituted H atoms of the methane nucleus. The compound 
thus formed, which bears the same relation to the phenyl methane 

58 



as alcohol does to methane, is called a carbinol. A carbinol is 
methyl alcohol in which one or more of the hydrogen atoms may 
have been replaced with an alkyl radical or a benzene ring. Thus : 




OH 

diphenyl carbinol 

It is next theoretically possible to attach amino groups to the ben- 
zene rings. Thus in the case of di-phenyl carbinol it is possible to 
obtain di-amino di-phenyl carbinol: 




NH2 



H.N 



Now this latter compound contains the necessary auxochrome 
groups; but it is not yet a dye. Xo carbinol is a dye, because it 
lacks a chromophore group. The carbinols are important in dye 
chemistry, however, because upon dehydration a rearrangement of 
the bonds in the molecule takes place giving the quinoid benzene 
ring, which as we have seen is a powerful chromophore. Thus: 



H.N 




NH.^H.N_/~\_C 




,NH+H.O 



Now this latter compound is the anhydride of a true dye base. 
Upon hydration it should theoretically become: 



H.N 




Such a compound could exist only in watery solution, 
only by its salts, the true dyes, as: 



It is known 



H.N 




Altho the theoretical compound given above is the true dye base, 
the carbinols are often known as carbinol bases of the phenyl 
methane dyes or are sometimes called leuco-bases or color bases. 
They are not bases in the chemical sense, however, as they do not 



59 



have basic properties. As stated above, they lack the chromophore 
group, and hence are colorless. 

1. DI-PHENYL METHANE DERIVATIVES 

The di-phenyl methanes are of practically no biological signif- 
icance. Only one deserves mention here. 

AURAMIN C. I. NO. 655 

Synonyms: Canary yellow. Pyoktaninum aureum. 

Pyoktanin yellow. 

CH3 CH3 

\N_/-\_C_/-\v_N/ 

/ \_/ I \_/ \ 

CH3 NHa CH3 

CI/ 

Altho of some use as a drug, auramin has little value in micro- 
scopic technic. It has been used by Fischel, however, in the vital 
staining of salamander larvae, and by Vinassa for staining plant 
sections.* 

2. TRI-PHENYL METHANE DERIVATIVES 

There are two groups of tri-phenyl methanes to concern us, the 
amino and the hydroxy derivatives. The former, which are much 
the more numerous, are very strongly basic, thanks to the amino 
groups, unless sulfonated like light green or acid fuchsin. The 
rosolic acid dyes, on the other hand, are hydroxy phenyl methanes, 
the amino groups being replaced by hydroxyl groups; they are 
therefore acid instead of basic dyes. 

There are likewise two subdivisions of the amino derivatives, 
the di-amino tri-phenyl methanes and the tri-amino tri-phenyl 
methanes. These two groups are derivatives respectively of: 
di-amino tri-phenyl methane 

H.N_/~\ H 

\_/\ / 
C 

H.N_/— \/ \/"~\ 
\_/ \_/ 

and tri-amino tri-phenyl methane, or pararosanilin. 

H.N /^\ H 




H.N /~\/ \/~X NH, 



The individual dyes of this series are substitution products of these 

*Literature References to the procedures mentioned in this chapter may be 
found on pp. 110 to 128 and 138 to 145. 

60 



two compounds and differ from one another in the number of 
methyl, ethyl, or phenyl groups introduced, and according to 
whether they are introduced into the amino groups or directly 
onto the benzene rings. 

a. Di- AMINO Tri-phenyl Methanes 

MALACHITE GREEN C. I. NO. 657 

Synonyms: Emerald green. New victoria green. Diamond green. 

Solid green. Light green N. 




_C 




{Absorption maxima: 616, [4^0] ) 

Malachite green is a rather weakly basic dye that has been used 
in the past for various histological purposes; as by V. Beneden for 
staining Ascaris eggs, by Petroff for staining erythrocytes, and by 
Maas as a contrast stain following borax carmine. Today it has 
very largely been replaced by methyl green; but it is now often 
used by botanists for staining host tissue in plants infected w4th 
fungi, according to the technic of Pianese (with acid fuchsin and 
martins yellow), which was originally applied to cancer tissue. 

BRILLIANT GREEN C. I. NO. 662 

Synonyms: Ethyl green. Malachite green G. 

This is a basic dye which is generally known in the form of the 
sulfate : 



CH3CH. SO.H / \_N 




CH2'CH3 

/ 



CH.CH3 



{Absorption maximum: 623.) 

The largest call for brilliant green at present is as an indicator 
in media for water analysis, according to the technic of Krum- 
wiede. It is also used for inhibiting the colon organism in stools; 
and is added to broth for the enrichment culture of the typhoid 
bacillus. 

61 



LIGHT GREEN SF, YELLOWISH C. I. NO. 670 

Synonyms: Light green 2G, 3G, 40, or 2GN. Acid green (with 
various shade designations). Fast acid green N. 

This is a derivative of brilUant green, which is sulfonated and is 
therefore an acid dye. 

0x12 'CH^ 

/ 
CH3CH2 /^\^N 

N_/~\_C I CH2_/^\_S03-Na 

/ \_/ l/~\_S03 \_/ 

NaS03_/'~\_CH, \_/ 

\_/ 

(Absorption maximum: 633.5.) 

Light green is a valuable plasma stain often used for staining 
tissues in contrast to iron haematoxylin, altho it fades badly if 
exposed to bright light. It is used by Benda in contrast to safranin 
as a cytoplasm stain for spermatozoa. In plant histology it is a 
useful cytoplasm and cellulose stain. 

b. Tri-amino Tri-phenyl Methanes (Rosanilins) . 

The simplest rosanilins are the dyes sold as basic fuchsin. This 
term seems to be somewhat loosely used to apply to two or three 
different dyes and to various mixtures of them. The dyes known 
as fuchsin differ from the methyl violets and other rosanilins in 
that the amino groups are not methylated or substituted in any 
other way. The fuchsins may, however, have methyl groups in- 
troduced directly onto the benzene rings instead of into the amino 
groups; and the different fuchsins vary from one another in the 
number of such methyl groups present. There are four primary 
compounds theoretically possible, namely with no methyl group, 
and with one, two, and three substituent methyl groups respective- 
ly. The types commonly encountered are listed below. 

Basic fuchsin is a very valuable stain, and is one of the most 
powerful nuclear dyes. It is also a stain for mucin, for elastic 
tissue, and for bringing out the so-called fuchsinophile granules. It 
is often used for staining the nuclear elements of the central ner- 
vous tissue. It is one of the most useful bacterial stains, particularly 
in the Ziehl-Neelson method for differentiating the tubercle organ- 
ism and thus diagnosing tuberculosis. As an indicator it is used to 
distinguish the typhoid organism from other closely related forms 
by means of the Endo medium, in which it is reduced to the color- 
less leuco-fuchsin by the use of sodium sulfite. This medium 
(which contains lactose) remains colorless in the presence of the 
typhoid organism, which does not attack lactose; but becomes 
colored in the presence of organisms like Bacterium coli which fer- 

62 




ment the lactose (a reaction that converts the leuco-fuchsin again 
into the dve fuchsin). 

PARA-FUCHSIN C. I. NO. 676 

Synonyms: Basic rubin. Pararosanilin. Para-magenta, 

H CI 

\l 

^= 

/ " 

H 

(A basic dye; absorption maximum about 539.) 

This dye is frequently sold as basic fuchsin. Investigation, in 
fact, shows that most of the stains sold under that name are really 
pararosanilin chloride. The acetate is also sometimes encountered. 
It is evident that for many purposes for which fuchsin is used, this 
is satisfactory; but a recent investigation by the Commission shows 
that it is ordinarily less desirable than the higher homologs, es- 
pecially rosanilin. 

ROSANILIN 

This compound is mono-methyl fuchsin, or triamino-tolyl- 
diphenyl-methane chloride. 




XH2 



{A basic dye; absorption maximum about 542.) 

It is not a textile dye, and is not found free from pararosanilin 
unless specially prepared. One stain company, it is found, has 
supplied it as "basic fuchsin, for Endo medium." For this par- 
ticular purpose, indeed, rosanilin hydrochloride proves to be the 
best adapted of any of the basic fuchsins; and, so far as it has been 
investigated, it is a very satisfactory stain for all purposes for 
which basic fuchsin is ordinarily used. 

BASIC FUCHSIN C. I. NO. 677 

Synonyms: Diamond fuchsin. Magenta. Rubin. Anilin red. 

Various shades occur, denoted by different designations. 

This dye, as furnished for textile purposes, is a mixture in about 
equal parts of pararosanilin and rosanilin. 

As the greater methyl substitution of the latter compound causes 
it to have a deeper shade than pararosanilin, the dye varies in 

63 



depth according to the amount of rosanilin present. These mix- 
tures are the various fuchsins known to commerce. In the course 
of the recent investigation of fuchsins, no basic fuchsin of this type 
has been found offered to biologists as a stain. Whether it would 
prove satisfactory for staining purposes still remains to be deter- 
mined. 

NEW FUCHSIN C. I. NO. 678 

Synonyms: Isonihin. Fuchsin NB. 

This compound is tri-methyl fuchsin, or triamino tritolyl 

methane chloride: 

CH3 

H CI /~\_NH. 

N_/^_C 




_ "\_NH. 

H 

CH3 ! 

CH3 

{A basic dye; absorption maximum about 5^4, 5J^5.) 

This dye is sometimes sold for a stain under the name of basic 
fuchsin, altho the most reliable companies sell it under its correct 
name. Recent investigations show that it may be admirable for 
all the staining purposes for which basic fuchsin is used. 

ACID FUCHSIN C. I. NO. 692 

Synonyms: Fuchsin 8, S.Y, SS, ST, or S III. Acid magenta. 

Acid rubin. 

{An acid dye; absorption maximum about 5^5.) 

This dye owes its acid character to the fact that it is a sulfonated 
derivative of basic fuchsin. Acid fuchsins are ordinarily rather 
complex mixtures. As there are four primary basic fuchsins pos- 
sible, according to the degree of methyl substitution, and as each 
may yield at least three different compounds on sulfonation, fully 
a dozen acid fuchsins are theoretically possible, and samples are 
hardly to be expected which are not mixtures of several. 

The generally accepted formula of one of the homologs present 

in acid fuchsin, namely the di-sodium salt of rosanilin trisulphonic 

acid, is: 

SOj-Na 

/ 
CH3 /— \_NH. 

\ IIV_/ / 

H2N_/^_C SO3 



NaSO, 




NH2 



64 



The bond connecting one of the sulfonic groups with an amino 
group attached to a different benzene ring is assumed to exist in 
order to account for the fact that altho only two of the sulfonic 
groups are neutralized Avith sodium, the compound acts as tho it 
has no free acid. In other words, it is a case of intramolecular salt 
formation. Now when the tri-sodium salt is formed, this bond is 
broken down, whereupon the quinoid ring disappears and the fol- 
lowing compound is produced: 

SOrXa 



H.X 




XH. 



XaS03 \_/ 

This compound, it will be seen, is a carbinol in structure, and as it 
lacks the quinoid ring it is colorless; but it is very readily converted 
into the di-sodium salt by the addition of acid, whereupon the color 
again appears. This property makes acid fuchsin of use as an 
indicator. The decolorized solution of acid fuchsin neutralized 
with sodium hydrate is called the Andrade indicator. It is used 
quite extensively in bacteriological work, because of the striking 
reaction when its color is restored by acid-forming bacteria. As an 
indicator to show hydrogen-ion concentration at all accurately, 
however, it is found to have much less value than the phthalein 
and sulphonphthalein dyes (see pp 83 to 86.) 

Acid fuchsin is a widely used plasma stain, which has also been 
recommended for a number of special uses. Among the best known 
are: the Van Gieson connective tissue stain, in which it is used with 
picric acid after haematoxylin to differentiate smooth muscle from 
connective tissue; the Ehrlich-Biondi stain, in which with methyl 
green and orange G it is employed in histology and for staining 
blood smears; and the Ehrlich tri-acid stain for blood, which is a 
"neutral" combination with orange G and methyl green. In plant 
histology it is used to stain the cortex, pith and cellulose walls; 
while the Pianese stain (with malachite green and martins yellow), 
originally applied to cancer tissue, is now used by plant pathologists 
in studying infected vascular plants. It is used with methyl green, 
bv Altmann, Benslev and Cowdrv as a stain for mitochondria. 
To the pathologist it is quite valuable as a constituent (with anilin 
blue and orange G) of the Mallory connective tissue stain. 

HOFFMAN VIOLET C. I. NO. 679. 

Synonyms : Dahlia. Iodine violet. Red violet. J^iolet R, RR, or J^RN. 

Hoffman violet is derived from fuchsin bv the introduction of 
methyl or ethyl groups (generally the latter) into the amino groups. 
Thus the tri-ethvl rosanilin has the formula: 

65 



CI 

I 

H 



/ 



/ 
"X^C 




/ 



H 



\/' 



N 



' / ■ 

N 

\ 
H 



{A basic dye.) 

The dyes known to commerce are mixtures of the higher and 
lower homologs of this series. The higher homologs, on account of 
the presence of ethyl groups and the higher molecular weight re- 
sulting therefrom are a very deep violet. 

Hoffman violet has been used by Ehrlich and by Unna for stain- 
ing mast cells; and by Juergens for staining amyloid, which stains 
red, while the cytoplasm is colored blue. 



METHYL VIOLET 



C. I. NO. 680 



Synonyms: Dahlia B. Paris violet^ Pyoktanin blue. 

Gentian violet. 

Various shades denoted: Methyl violet 3R, 2R, R, B, 2B, 3B, 
BBN, BO, 3V. 

The various dyes denoted methyl violet are mixtures of tetra-, 
penta-, and hexa-methyl pararosanilin : 



H CI 

\l 

/ " 
H 



CH, 



CH3 
C CH, 

N 

_/ \ 

CH3 

tetra-methyl pararosanilin 




CH3 CI 




CH, 



penta-methy] pararosanilin 
66 





CH3 




/ 


/ \_ 


N 


CH, CI /\ / 


\ 


\ / 


CHj 


N / \_C 


CHj 


/ \ / \ 


/ 


CH3 \/^V- 


N 


\ / 


\ 




CH, 


hexa-methyl pararosanilin 


(crystal violet) 





{Basic dyes; absorption maxima: 583-58 J^ in 90% alcohol.) 

In the case of these compounds, as in the case of other series of 
homologs differing in extent of methylation, the shade is deepened 
by the introduction of each methyl group. Hence the various 
mixtures known to the trade as methyl violet vary from reddish to 
bluish violets according to the relative amounts of the more and 
less completely methylated compounds present in the mixture. 
This is the significance of the various shade designations listed 
above, R's indicating the reddish shades, and B's the bluish shades. 
Of these various shades the bluer ones seem to be best for biological 
purposes, methyl violet 2B having been found satisfactory for 
practically all purposes for which methyl or gentian violet is 
ordinarily called for. This indicates that the biologist requires the 
higher homologs in this group. Now the most completely methy- 
lated methyl violet is the hexa-methyl compound, which is easily 
obtained pure and is known to the trade as crj^stal violet. This 
dye, therefore, appeared very interesting to the Commission and 
has been given considerable investigation. 

CRYSTAL VIOLET C. I. NO. 68 1 

Synonyms: Violet C, G, or 7B. Hexamethyl violet. Methyl violet 

lOB. Gentian violet. 

(A basic dye; absorption maximum about 591.) 

This dye is hexa-methyl-pararosanilin, whose formula is given 
above as one of the components of methyl violet. 

The Commission has made as careful an investigation of this 
dye as of any other and has become very enthusiastic over it. 
Methyl or gentian violet is of chief value to the biologist as a 
nuclear or chromatin stain, having many histological and cyto- 
logical applications, the one for which it is most commonly used at 
present being the Flemming triple stain in which it is employed 
with orange G and safranin — a technic which gives a very high 
degree of differentiation. It is also used for staining amyloid in 
frozen sections of fresh and fixed tissue, and for staining the plate- 
lets in blood; while it is much used by the Weigert technic for stain- 

67 



ing fibrin and neuroglia. The bacteriologist also finds it a useful 
stain and probably purchases more at the present time than all 
other biologists together; the chief bacteriological use is in the 
Gram technic for distinguishing between different kinds of bac- 
teria. A further more recent use is in bacteriological media for in- 
hibiting the growth of Gram-positive organisms, due to its selective 
bacteriostatic action. 

The Flemming and Gram stains have seemed the most delicate 
procedures for which it is used; so they have been given the most 
careful study. In the case of the Gram stain it was discovered that 
there are a score or more different procedures all referred to by the 
name "Gram" stain, and a study was made of all the methods that 
were found (see Hucker and Conn 1923). The result of the in- 
vestigation is to conclude without reservation that crystal violet 
may be substituted for gentian violet in both the Gram and Flem- 
ming technics, and probably for gentian or methyl violet in any of 
the bacteriological or histological methods for which either stain 
is designated. If crystal violet can be used in all cases, the ad- 
vantage is obvious; for it is a definite chemical compound, while 
methyl and gentian violet are both variable mixtures. 

It is of interest to note that in the literature of microscopic tech- 
nic crystal violet has been specified instead of gentian violet for 
some special procedures. Worth noting is Benda's crystal-violet- 
alazirin method for staining chondriosomes, and its modifications 
by Meves and Duesberg; and also its use in combination with 
erythrosin by botanists for staining lightly lignified walls, in which 
technic it proves more uniform than gentian violet. 

GENTIAN VIOLET 

A poorly defined mixture of violet rosanilins is well-known to 
biologists under the name gentian violet. The name is not used at 
present in the dye or textile industries, however, and for this reason 
the dye is not listed in dye indexes. It apparently applies to a 
certain mixture containing about half dextrin and half dye, the 
dye being a methyl violet, that is a mixture of crystal violet with 
lower homologs of the same series. The statement has been made 
and often repeated in biological literature that gentian violet is a 
mixture of crvstal and methvl violet; but the looseness of the state- 
ment is evident when it is realized that crystal violet is a component 
of all the deeper shades of methyl violet. It is possible that before 
the war gentian violet did represent a fairly constant mixture, but 
there seems to be some doubt even on this point. It is certain that 
since the war each company has used its own judgment as to what 
to furnish when gentian violet is ordered. One company admits 
furnishing crystal violet unmodified under this name; another 
claims to be supplying penta-methyl-pararosanilin for gentian 
violet; another apparently has been mixing crystal violet and 

68 



methyl violet 2B together with an equal weight of dextrin. Yet in 
actual test, none of these preparations proves any better than 
crystal violet or methyl violet 2B, unless it be possibly in inhibit- 
ing the growth of Gram-positive bacteria. Unfavorable reports 
of crystal violet for this purpose have been received; but tests 
of several samples of crystal violet now on the market show 
them to be bacteriostatic to a higher degree than the sample of 
pre-war gentian violet with which they were compared. 

Under the circumstances the Commission has faced a difficult 
problem in trying to standardize gentian violet. The question has 
been whether to recognize the name at all, or to approve some par- 
ticular dye or mixture of dyes of this group as gentian violet. The 
former course was almost impossible because of the wide demand 
among biologists for gentian violet under that name. The second 
course (unless considerable latitude be recognized) would be en- 
tirely arbitrary, inasmuch as no information is available to show 
which members of this group of dyes are especially needed in hist- 
ology or bacteriology. Accordingly the Commission has finally 
defined gentian violet as either penta-methyl or hexa-methyl para- 
rosanilin, or else a mixture of methylated pararosanilins composed 
primarily of the two compounds just named and having a shade at 
least as deep as that recognized in the trade as methyl violet 2B. 
Gentian violet, must, moreover, prove satisfactory in actual use 
as determined by the tests proposed by the Commission (see 
specifications page 135). This definition will not exclude anything 
now sold as gentian violet except those that do not prove satis- 
factory in performance. At the same time the Commission does 
not wish to give official standing to gentian violet and recommends 
that crystal violet be ordinarily ordered in its place. 

METHYL GREEN C. I. NO. 684 

Synonyms: Double green. Light green. 
{A basic dye; absorption maximum about 633.8) 

Methyl green is crystal violet into which a seventh methyl group 
has been introduced by the action of methyl chloride or methyl 
iodide upon it, forming the compound:* 




*This ordinarily occurs in trade as a zinc double salt. 

69 



As the seventh methyl group is very loosely attached, there is 
always some methyl violet present, either because it is not all com- 
pletely converted into the higher homolog or because it has broken 
down again. It has been stated that to obtain free methyl green 
the commercial dye should be shaken in a separatory funnel with 
amyl alcohol or chloroform, which dissolves the methyl violet. As 
a matter of fact, however, pure methyl green may not be always 
desired by the biologist, as the dye owes part of the metachromatic 
properties for which it is prized to the presence of small amounts of 
the violet compound. 

Methyl green is at present one of the most valuable nuclear 
stains known to the histologist, and is widely used as a chromatin 
stain by the cytologist. On the other hand it has been used by 
Galeotti as a cytoplasm stain following acid fuchsin and picric 
acid. In the Ehrlich-Biondi technic it is used to stain nuclei in 
contrast to acid fuchsin; while Bensley employs it to stain chroma- 
tin in contrast to acid fuchsin which stains the mitochondria. It is 
an ingredient of the Ehrlich triacid mixture (with orange G and 
acid fuchsin) for staining blood smears. Botanists find it a valu- 
able stain, combined with acid fuchsin, for lignified xylem. One of 
its most valuable uses today is in the Pappenheim stain, in which 
it is combined with pyronin and used for staining the gonococcus 
and mast cells as well as by Unna in studying chromolysis. It is 
also a useful chromatin stain for protozoa, and is employed in weak 
acetic acid solution for staining fresh material beneath the cover- 
glass. 

When the foreign supply of dyes was first shut off, this stain 
proved one of the most difficult to obtain in satisfactory quality, 
largely due to the looseness with which the seventh methyl group 
is attached and the resulting instability of the compound. At first 
certain green dyes of an entirely different nature were furnished, 
but as soon as an investigation of the dye was begun manufac- 
turers proved perfectly able to produce methyl green; the difficulty 
came in obtaining the right degree of purity. Samples were finally 
furnished so pure that they lacked completelj^ the necessary meta- 
chromatic staining quality; and it proved necessary to add a certain 
small percentage of the violet dye to obtain the proper results. This 
problem seems to have been solved at present and satisfactory 
methyl green is available. The chief problem now is to standardize 
it. With other stains this can ordinarilv be done on the batch 
basis, approving some batch large enough to meet the demand for 
a period of years. With methyl green this cannot safely be done, 
on account of its instability. Hence large batches are impractical; 
and the stain ought to be sold with the caution that the dye does 
not keep indefinitely without change. That this is not generally 
realized is shown by the fact that when a certain company recently 
announced for sale a supply of German stains imported before the 
war and kept on their shelves since then, one laboratory ordered a 

70 



pound of methyl green. For the purposes for which this stain was 
desired this batch may be satisfactory; but the user can hardly 
count on its being the same as it was when imported nor on its 
remaining unchanged until used up. 



IODINE GREEN 

Synonym: Hoffmann s green. 



c. I. NO. 686 



This dye is closely related to methyl green, the generally accept- 
ed formula being: 



CHj CI 

\l 
N_ 

/ " 
CH, 



CI CHj 

1/ 
/-\_N-CH3 

/V_/ \ 
/ CH3 

.=C CHj 

./ \ ^ / 

\ CH3 

CHj 



{A basic dye.) 

Iodine green is a nuclear or chromatin stain which has selective 
properties that make it of value in certain special procedures. It 
is used by Ciaccio for nervous tissue in combination with acid 
fuchsin and picric acid; and by Lefas as a blood stain in combina- 
tion with acid fuchsin. It is used by Zimmermann with basic 
fuchsin for staining chromatin in plant tissue; while together with 
acid fuchsin it is occasionally used by botanists for staining lignified 
xylem. It is also used for staining mucin and amyloid, having the 
property of giving the latter a red instead of a green color. 



SPIRIT BLUE 



C. I. NO. 689 



Synonyms: Gentian blue. Anilin blue, alcohol soluble. Night blue. 

Lyons blue. Paris blue. 

This is a mixture of di-phenyl rosanilin 




CH, 



71 



and tri-phenyl rosanilin: 



CI 

I 

N_ 

T 

H 




_C 



/ 




H 

I 

N 



\_/ 




{Basic dyes; absorption maximum of spirit blue 2R 
about 581 in alcohol.) 

It is used in contrast to carmin in staining embryonic tissues; it 
brings out growing nerve fibers well. 



METHYL BLUE C. I. NO. 706 

Synonyms: Cotton blue. Helvetia blue. 



NaSO, 



H 

I 

N 




SO.Na 



{An acid dye; absorption maximum about 607.) 

On account of the sulphonic groups, this dye is strongly acidic 
and makes a good counter-stain. It is used by Mann with eosin 
for staining nerve cells; and by Dubreuil, combined w4th picric 
acid, in contrast to a red nuclear stain such as carmin or safranin. 



ANiLiN BLUE, w. s. (i.e.. Water soluble) c. i. no. 707 

Synonyms: China blue. Soluble blue 3M or 2R. Marine blue. 
Cotton blue. Water blue. Berlin blue. 

This dye is a mixture of the tri-sulphonates* of tri-phenyl para- 
rosanilin (C. I. 706) and of di-phenyl rosanilin. The latter is: 



•"The location of the sulphonic groups is uncertain. 



72 



NaSO., 

ch/ 




SO,Xa 



(An acid dye; absorption maximum of water blue 2B 

about 5Jf.6.5.) 

It is a widely used histological stain, having valuable counter- 
staining properties. It is also of use as an indicator, due to the dis- 
appearance of the color upon complete neutralization, as in the 
case of acid fuchsin. As an indicator, however, it has the disad- 
vantage that the blue color is but slowly restored upon addition of 
acid. 

Its chief histologica uses are : by Stroebe and Huber as a cyto- 
plasm stain preceding safranin; by Galli for axis cylinders; fre- 
quently by botanists as a contrast for safranin in vascular plant 
tissue, or for magdala red in algae; and very widely by pathologists 
in the Mallory connective tissue stain, in which it is combined with 
orange G and acid fuchsin; by Unna in contrast to orcein for stain- 
ing epithelial sections, and in studying the process of chromolj^sis. 

(c) Hydroxy Phenyl Methanes (Rosolic Acids) 

The rosolic acid dyes, as stated above, are tri-phenyl methane 
derivatives in which the amino groups of the rosanilins are re- 
placed with hydroxyl groups, thus giving them acidic instead of 
basic character. The compounds of this group are not very im- 
portant as dyes and are scarcely used as stains. The greatest 
interest of the biologist in them is due to their use as indicators, 
since in acid solution the quinoid ring disappears and the compound 
becomes colorless, while alkali changes it back to the colored form. 
Thus: 

H /—\ OH 



HO 




OH 



+2XaOH = 



leuco-rosolic acid 



+ sodium = 
hydroxide 




_C 



+2H.0 




disodium rosolate 



7S 



There is considerable confusion in the nomenclature of these 
dyes, as the names employed may be used in a strict chemical sense 
or in a looser sense in practice. Chemically there are two rosolic 
acids, which are related just as are rosanilin and pararosanilin. 
Pararosolic acid differs from pararosanilin, only in having hydroxyl 
groups in place of the amino groups : 

H.N /~\ /^ NH. HO /^\ /—\ OH 



OH 




.NH^ 
HO 

Pararosanilin carbinol 




leuco-pararosolic acid 



Rosolic acid, on the other hand, is a mono-methyl derivative, and 
bears the same relation to rosanilin : 



H.N 



CH, 




NH. 



HO 




OH 



c / c 

/ \/~\_NH2 CH 

HO \_/ 

Rosanilin carbinol leuco-rosolic acid 

Now the dye to which the name rosolic acid or aurin is generally 
given in practice is a mixture consisting of both rosolic acid and 
pararosolic acid together with other closely related compounds. 
This dye is: 

AURIN OR ROSOLIC ACID C. I. NO. 724 

A mixture of rosolic acid and pararosolic acid, with oxidized and 
methylated derivatives of the latter. This product is of consider- 
able use as an indicator. 

Corallin yellow is the name given to its sodium salt. 

No other dyes of this group have biological use. Two others 
perhaps deserve mention: 

CORALLIN RED C. I. NO. 726 

Synonym: Aurin R. 

Apparently a compound dye, the pararosanilin salt of pararosolic 
acid. 



CHROM VIOLET 

A carboxyl derivative of pararosolic acid : 

COONa 



0_/~X— 



C. I. NO. 727 




/ 
COONa 



COONa 



74 



CHAPTER VII 



THE XANTHENE DYES 

THE group of colored compounds known as xanthese dyes com- 
prises a number of basic and acid dyes and quite a series of 
indicators. In fact, the most valuable indicators known to the 
chemist fall in this group. They are derivatives of the compound 

xanthene : 

O 




C 

/ \ 
H H 



1. THE PYRONINS 

The pyronins are methylated di-amino derivatives of xanthene. 
They are closely related to the diphenyl methanes and are some- 
times classed with them, as they have a carbon atom attached to 
two benzene rings, and show the same tendency toward quinone 
structure. Their formula, on the other hand is like that of the 
oxazins except that the nitrogen of the central ring is replaced by 
a methenyl (CH) radical. Like the oxazins, the atomic grouping 
may be assumed to be in either the paraquinoid or the ortho- 
quinoid form, thus: 



H 

/ 
H,N_/\_0_/\_N— H 

CI 



H.N 



or 




NH. 



Another arrangement of the atoms is possible in which no quinoid 
ring exists, namely: 



H.N 



CI 

I 

o 



NH. 



"C" 

I 
H 



This latter form might also be assumed for the oxazins and thiazins 
as well, and this type or formula is frequently used for the azins; 
but the xanthene dyes are more often represented in this form. If 
this formula is adopted the quinoid ring cannot be accepted as 



75 



their chromophore. For this reason one of the quinoid formulae 
seems preferable; and for the sake of uniformity the paraquinoid 
form will be given in the following pages. It must be remembered, 
however, that the other formulae are equally admissable; and it is 
possible that the compound occurs in two or even all three of the 
different forms. 



PYROXIN G 



c. I. NO. 739 



CH, 



CH, 



\ 

I 

/ 



N 




CH3 

/ 
_X— CH3 

"I 
CI 



{A basic dye; absorption maximum about 5^5.) 

This dye finds its principal use as a stain in the Pappenheim 
combination, where it is employed with methyl green for staining 
basophile elements, especialty the mast cells, and for staining the 
gonococcus in smears of pus. It is also used sometimes as a counter 
stain in the Gram technic for bacteria; and by Ehrlich as a com- 
ponent of certain "neutral" stains. Since the war it has proved 
difficult to obtain this dye in America except by importation, as 
the intermediate from which it is manufactured is very difficult to 
prepare and is not produced in this country.* 



CH.CH 



3 ^^^2 



\ 



N 



PYRONIX B C. I. NO. 741 

CH.CH3 
O /\_N— CH. •CH3 



CH.CH 



3 '-'^^2 




{A basic dye; absorption maximum about 550.) 

This dye differs from pyronin G only in that it is an ethyl in- 
stead of a methyl derivative. As a result it is very slightly deeper 
in shade but has almost identically the same staining behavior. 
Investigations recently carried on by the Commission indicate that 
it can replace pyronin G in the Pappenheim stain and probably in 
all its other uses. This is very encouraging, for it is much more 
easily prepared and a very satisfactory product of American 
manufacture is now available. 



*For literature references to the procedures mentioned in this chapter see 
pp 110 to 128 and 138 to 145. 



76 



2. THE RHOD AMINES 

The rhodamines are similar to the pyronins except that there is 
a third benzene ring attached to the central carbon atom and 
attached to this ring is a carboxyl group in the ortho position. 
This latter group, altho of acid tendency, does not counteract the 
basic action of the amino groups, so the dyes are basic in character. 
Only one of them is of any significance to the biologist, namely: 



CH.CH 



3 ^^^2 



RHODAMINE B C. I. NO. 749 

Synonyms: Rhodamine 0. Brilliant pink. 

CH2CH3 

O /\_N— CH.CH 

"I 
CI 

GH3'CH2 

I 

COOH 




{A basic dye; absorption maxima about 556.5^ [519]) 

A rhodamine, probably the above dye, has been used by Gries- 
bach with osmic acid to fix and stain blood simultaneously; by 
Ehrlich as a component of "neutral" stain mixtures; by Rosen 
for histological work in contrast to methylene blue; and by others 
in contrast to methyl green. 

A somewhat different dye, knoTvn as Rhodamine S (C. I. No. 
743) has been mentioned in the same connection and may have 
been used for some of the above mentioned purposes. It is not a 
true rhodamine, however, but belongs to a closely related group of 
compounds, the succineins; for it does not have the three benzene 
rings, the radical CgH^COOH being replaced by C2H4COOH. 

In practice the rhodamines are prepared not from xanthene but 
by the condensation of two molecules of meta-amino phenol, 



NH. 




OH 



with one of phthalic anhydride : 




This shows their close relation to the next group of dyes, namely 
the fluorane derivatives, which as will be seen are also prepared 
from phthalic anhydride. In fact these two groups of dyes, acid 
and basic respectively, are related in exactly the same way as the 
rosolic acids and the rosanilins, the one group having hydroxyl 
radicals where the other has amino groups. 

3. FLUORANE DERIVATIVES 

Fluorane is not a dye, but is a very important compound in dye 

chemistry. It is a derivative of phthalic anhydride, and contains 

a xanthene ring (five C atoms and one O atom) as well as a lactone 

ring (four C atoms and one O atom) besides three benzene rings; 

thus : 

O 




c 

I "CO 



The fluorane dyes are derivatives of this by the introduction of 
hydroxyl groups into two of the benzene rings at the para position 
to the central carbon atom and the further introduction of halogen 
atoms at various positions in all three benzene rings. 

It proves convenient here to class these compounds with the 
xanthene dyes. They may, however, be equally well considered 
tri-phenyl-methane dyes, as can be seen by a glance at the formula 
of any of them; in fact they are generally so considered by the 
chemists. To the biologist they stand in a distinctly different class 
from the tri-phenyl-methanes ; and for that reason are treated here 
instead of in the preceding chapter. 

FLUORESCEIN C. I. NO. 7 66 

Synonym: Uranin. 

This is the simplest of the fluorane dyes, and is the mother sub- 
stance of the eosins. The composition of its sodium salt is: 

NaO 

O /\-0 



'C= 



COONa 



\/ 



(An acid dye; absorption maximum about 4-90.) 

78 



EOsiN, Y (i.e., yellowish) C. I. NO. 768 

Synonyms: Water soluble eosin. Eosin W or WS. 

Various shades denoted: Eosin G, Y extra, S extra, J extra, 
B extra, GGF, 3J, 4J, KS, DH and JJF. 

{An acid dye; absorption maximum about 516.) 

This dye is typically tetrabrom fluorescein : 

Br Br 



NaO 




COONa 



but the mono- and di-brom derivatives are also known and fre- 
quently occur in eosin. This affects the shade, as the more bromine 
present the redder the dye. It is plain that various mixtures of 
these compounds are on the market; but it has not yet been deter- 
mined which are more suitable for biological purposes. Consider- 
ably more work on eosin is needed than has been done at the 
present time. From the name "water soluble eosin" it is often 
assumed that this dye is not soluble in alcohol. This is not true, 
however. 

Yellowish eosin is one of the most valuable plasma stains known. 
It is used in various technics for staining the oxyphile granules of 
cells (i.e., the granules having special affinity for acid dyes); these 
cell elements, in fact, being often called eosinophile granules be- 
cause their presence was first recognized thru the use of this dye. 
It is often employed as a counterstain for haematoxylin and the 
green or blue basic dyes; as for example by Mallory with methy- 
lene blue,* and by List with methyl green. It is used by Mann 
mixed with methylene blue as a tissue stain; and by Teichmuller 
for staining sputum before staining with methylene blue. At the 
present time one of the uses for which it is in greatest demand is 
as a blood stain in the technic of Romanovsky, with its various 
modifications, in which it is combined with methylene blue to 
form a "neutral" stain. 

METHYL EOSIN C. I. NO. 769 

Synonym: Eosin, alcohol soluble. 

This is the methyl ester of yellowish eosin, the sodium salt of 
which is: 



*See, however, the statement about this technic below under phloxine (p. 82) 

79 





Br 




Br 




NaO 


\/\. « A-« 


Br 








Br 



COOCH3 

(An acid dye; absorption maxima about 520, [It.85.5] ) 

ETHYL EOSIN C. I. NO. 770 

Synonyms: Eosin, alcohol soluble. Eosin S. 
This is similar to methyl eosin, but is the ethyl ester: 

Br Br 

I 



NaO 



Br 




Br 



"COOCH.CH3 



{An acid dye; absorption maxima about 523.5, [Ji-87] ) 

An alcohol soluble eosin, either this dye or the preceding, is a 
valuable counterstain after Delafield's haematoxylin in general 
animal histology. 



EOSIN, BLUISH 



C. I. NO. 771 



Synonyms: Eosin BN, J5, BW, or DKV. Saf rosin, Eosin scarlet 
B or BB. Scarlet J, J J, V. Nopalin G. Caesar red. 

This is a dibrom derivative of dinitro-fluorescein. 

Br Br 

NaO I I 

O /\_0 



NO2 



NO, 
"COONa 



{An acid dye; absorption maxima about 521.5, {Ji-86\ ) 

80 



ERYTHROSIN, YELLOWISH 



C. I. NO. 772 



Synonyms: Erythrosin R or G. Pyrosin J. Dianthine G. lodo- 

eosin G. 

This is a fluorescein in which there are two substituent iodine 
atoms instead of four bromine atoms as in yellowish eosin. 



O 



_0 



I 
COONa 



{An acid dye; absorption maximum about 510.5.) 



ERYTHROSIN, BLUISH 



C. I. NO. 773 



Synonyms: Erythrosin B. Pyrosin B. Eosin J. lodo-eosin B. 

Dianthin B. 

This is the tetraiodo compound corresponding to the tetrabrom 
compound of typical eosin. 

I I 

NaO I I 

O /\_0 




COONa 



{An acid dye; absorption maximum about 524--) 

Erythrosin has some use as an indicator. It is also em- 
ployed as a contrast stain for haematoxylin and certain blue and 
violet nuclear stains. Held uses it, preceding methylene blue, as 
a plasma stain for nerve cells. It is employed by Winogradsky for 
staining bacteria in soil. For these purposes probably the tetra- 
iodo compound (i.e., erythrosin bluish) is desired; but the litera- 
ture is vague on the subject. 

A sample of erythrosin of pre-war origin that was labeled Mag- 
dala red has been examined by the Commission. This mislabeling 
undoubtedly explains Chamberlain's results already mentioned 
(page 57) in staining algae. Chamberlain, it will be recalled, was 
able to obtain good results with a low priced product called Mag- 
dala red but not with the high priced stain called Magdala red 
echt. 



81 



PHLOXINE C. I. NO. 7 74 OF 778 

Synonyms: Erythrosin BB. New pink. 

(An acid dye; absorption maximum of C. I. No. 778 about 537.5.) 

If fluorescein is prepared from dichlor or tetrachlor-phthalic 
acid, instead of from simple phthalic acid, its derivatives have a 
deeper, more pleasing shade than the ordinary eosins. Of these 
derivatives, phloxine contains four bromine atoms and thus cor- 
responds to Eosin Y. It contains two or four chlorine atoms ac- 
cording to whether it is prepared from the dichlor- or the tetra- 
chlor-phthalic acid; and there are two Colour Index numbers cor- 
responding to these two compounds, the former being No. 774, the 
latter No. 778. It is uncertain which is desired by the biologist. 

The dichlor compound is : 

Br Br 

NaO I I 

O /\_0 



Br I Br 

Cl_/\_ 

COOXa 



CI 



Unna uses phloxine in combination with several other acid dyes 
in studying the process of chromolysis. The dye has seldom been 
specified for biological work; yet there is reason to believe that it is 
a more valuable stain than anyone has realized in the past, and that 
it has frequently been used under other names. 

Chamberlain (19'24 page 58) mentions having used it successfully 
in place of Magdala red in staining algae. His original technic 
called for Magdala red; but true Magdala red does not serve his 
purposes. Inasmuch as erythrosin (see above) was evidently sold 
in the past as Magdala red and Chamberlain can duplicate his 
original results with phloxine, the chances are that some of the 
Magdala red formerly available was either phloxine or else that 
phloxine and erythrosin give similar results by Dr. Chamberlain's 
technic. 

It has, furthermore, been found that no sample of eosin at 
present available of either domestic or foreign origin works in the 
well-known Mallory eosin-methylene-blue stain ; but after investi- 
gating both phloxine and rose bengal. Dr. Mallory reports the 
former to be "the best eosin I have yet found for use in the 
eosin-methylene blue stain for parafin sections of tissues fixed 
in Zenker's fluid."* Here again is a case where phloxine apparently 
was obtained before the war under an incorrect name and the incor- 
rect name used in the publication of a well-known technic. 

*Quoted from personal letter. 

82 



ROSE BENGAL C. I. NO. 777 OF 779 

Various shades denoted: Rose bengal B, 2B, 3B. 

{An acid dye; absorption maximum of C. I. No. 779 about 5^8.) 

This is a second derivative of di-chlor or tetra-chlor fluorescein, 
and corresponds to erythrosin, as it contains four iodine atoms. As 
in the case of phloxine, there are two similar dyes, the di-chlor 
compound (So. Ill) and the tetra-chlor compound (Xo. 779); it 
is not certain which is desired in microscopic work. The former 
has the formula 



NaO 



O 



C 



_0 



I I 

ci_/\_ 

COONa 

•CI 

This dye has a pleasing deep pink color; and altho an acid dye 
it proves to have considerable affinity for bacterial protoplasm, 
if used in carbolic acid solution, and to have good selective prop- 
erties when used as a bacterial stain. It has recently been recom- 
mended by the author for staining bacteria in soil suspensions. It 
has also been used as a cytoplasm stain following haematoxylin. 



4. PHEXOLPHTHALEIN AND THE SULPHOXPHTHALEINS 
A phthalein is a compound of phthalic acid : 

COOH 



I 



COOH 



or rather of phthalic anhydride: 




CO 



CO 



o 



with phenol or a phenol derivative. If phthalic acid is heated 
with phenol and sulfuric acid it combines with two molecules of 
the latter and forms phenolphthalein. In the same way, a sul- 
phonphthalein is a compound of ortho-sulpho-benzoic acid: 



SO3H 



COOH 




83 



and phenol or a phenol derivative. These compounds, altho some- 
times behaving as dyes, are not used as dyes or stains, but as in- 
dicators. For this purpose the members of the group are very 
valuable. 

Phenolphthalein, altho not used as a dye, is colored and is ap- 
parently capable of salt formation. In acid solutions it is colorless, 
and is assumed to have the formula : 

HO OH 





o 



I I c = o 

\/ 

Upon neutralization the alkali is believed to attach itself to the 
CO-group, which breaks the five-sided ring (the lactone ring) and 
causes one of the benzene rings to take on quinoid form, thus: 




H0_/\ /\_0 

I l_ =1 I 
\/ C \/ 

_ COOXa 

\/ 

With this change, the red color of the compound appears, but dis- 
appears again if the solution is made acid so as to destroy the quin- 
oid structure. This makes the compound a very valuable indi- 
cator. 

The sulphonphthaleins are not commercial dyes, altho capable 
of acting as acid dyes. Their real value is as indicators. Quite a 
long series of them has been prepared, which in general show their 
deepest color in alkaline solutions and turn yellow on the addition 
of acid. Fortunatelv nearlv everv one of them has a different 

*/ t t-' 

point in the hydrogen-ion scale at which its color change begins, so 
that between them they indicate very accurately the hydrogen- 
ion concentration of solutions of any reaction ordinarily encoun- 
tered. Some of them, such as thymol-sulphonphthalein (thymol 
blue), show two colors besides yellow, one in strong acid solutions 
and the other in strong basic solutions, while in solutions near the 
neutral point they are yellow. That these color changes are due to 
alterations in the structure of the molecule, such as the disap- 
pearance and reappearance of the quinoid ring, is generally as- 
sumed; but in the case of these compounds the relation of structure 
to color is complicated and has not yet been worked out to general 
satisfaction. 

84 



Phenol-sulphonphthalein {^phenol red) has the formula : 

HO OH 

\/\ /\/ 

c_ 
/ o 




so, 

\/ 

Among the other important sulphonphthalein indicators: Brom 
cresol purple is di-brom-ortho-cresol-sulphonphthalein: 

CH3 CH3 

HO I I OH 




Br C_ Br 

I O 



"SO2 



Thymol blue and Bromthymol blue are thymol-sulphonphthalein 
and di-brom-thymol-sulphonphthalein respectively; thus: 



CHj CH3 CH3 CH, CH3 CH3 CH3 CH 



3 



CH CH CH CH 

HO I I OH HO I I OH 

. '\ /\/ \/\ /\/ 

11 II and 11 





I Br I \ / I Br 

CH3 C_ CH3 CH3 C_ CH3 

/ o / O 



I I so, \ \ so. 

The formulae are all so similar that there is no need of giving the 
others. It is enough to state that brom phenol blue is tetra-brom- 
phenol-sulphonphthalein ; cresol red is ortho-cresol-sulphonphtha- 
lein ; brom cresol green is tetra-brom-meta-cresol-sulphonphthalein ; 
chlor phenol red is di-chlor-phenol-sulphonphthalein; brom phenol 
red is di-brom-phenol-sulphonphthalein; while brom-chlor phenol 
blue is di-brom-di-chlor-phenol-sulphonphthalein. 



85 



CHAPTER VIII 
COMPOUND DYES 

THERE are two ways in which dyes may be compounded. In 
the first place it is possible to mix mechanically any two dyes, 
and if they are of different colors with different selective powers, 
double staining effects may be procured. In the second place, it is 
often possible to form a chemical union between two dyes and thus 
to obtain an entirely new compound which may have quite strik- 
ing staining properties. It is such compounds as these, rather than 
simple mechanical mixtures, that are ordinarily referred to as 
compound dyes. 

The simple anilin dyes, it will be recalled (see Chapter II), owe 
their properties as dyes or as biological stains to the basic or acidic 
character of the dye molecule. Those parts of the protoplasm which 
are acid in nature (e.g., chromatin) tend to react with the basic 
dyes and to be colored with them; while those which are basic 
(e.g., cytoplasm) react similarly with the acid dyes. (This, to be 
sure, is not the whole theory of staining, as the process is quite 
complex and involves physical and mechanical factors as well ; but 
it serves to illustrate the difference between the two kinds of stains.) 
Now, as already explained, the dyes are not used as free acids or 
free bases; but rather as sodium or potassium salts of the acid 
dyes, and as chlorides (or salts of some other colorless acid) of the 
basic dyes. 

It is well known that when two salts, such as sodium chloride 
and ammonium nitrate, are mixed in solution there is an inter- 
change of ions, and the resulting solution, when it reaches equi- 
librium, contains not only the original salts but also the four free 
ions and the two alternate compounds as well, in this case sodium 
nitrate and ammonium chloride. Now if one of these two new 
compounds happens to be insoluble, as silver chloride for example, 
which would have been formed if silver nitrate had been substi- 
tuted for ammonium nitrate, it is thrown out of solution and equi- 
librium is not reached until the solution is free (or at least practi- 
cally free) from the two ions which are insoluble in combination. 
In the same way, when a sodium salt of an acid dye and a chloride 
of a basic dye are mixed in solution, there is a similar tendency for 
the ions to interchange. Ordinarily the dyes are weaker acids and 
bases than the chlorine and sodium ions respectively; and if the 
compound dye formed were soluble in water there would be little 
chance for much of it to be produced. As a matter of fact, however, 
it is generally insoluble and is therefore thro\\Ti out of solution; 
hence the compound dye can be formed in considerable quantitj'^. 

Now compound dyes of this sort are sometimes referred to as 
neutral dyes or neutral stains. This terminology, of course, does 

86 



not indicate that they are neutral in reaction any more than do the 
corresponding terms acid and basic dyes. A dye chemist, in fact, 
uses the term neutral dye in an entirely different sense; but it is 
frequently employed by biologists, especially by Ehrlich, to refer 
to the basic dye salts of dye acids. In this chapter these compound 
dyes will be called neutral stains, as this latter term is not employed 
by dyers or dye chemists in any sense, and is unlikely, therefore, to 
be misunderstood. Erhlich also uses another term to apply to 
some of these compounds, namely "tri-acid dyes." He uses this 
term on the assumption that, while in the ordinary basic dyes only 
one of the three affinities of the dye for acid is satisfied, it is pos- 
sible to satisfy all three and in this wav to saturate the basic dve 

«. »-■ t, 

with acid. This assumption of his seems to be incorrect; but 
Ehrlich's "tri-acid stain" (see below) is so well-known that an 
explanation of the term is necessary. 

It is possible to obtain endless variety of such dyes; but in prac- 
tice only a certain number of them have proved useful. Among 
the basic dyes the most suitable for this purpose are methylene 
blue and the rosanilins (which act as strong ammonium bases); 
among the acid dyes eosin and the sulphonic acids (e.g., orange G 
and acid fuchsin). 

Altho the neutral stains are insoluble in water, they are soluble 
to a greater or less extent in excess of either the acid or the basic 
dye. Thus if a watery solution of acid fuchsin is neutralized by 
adding drop by drop a watery solution of methyl green, there is at 
first no precipitation, because the methyl green salt of acid fuchsin 
is kept in solution by the excess of acid fuchsin. After the proper 
amount of methyl green has been added, however, and the mixture 
has stood long enough for the reaction to take place, the neutral 
dye is precipitated and the solution becomes nearly colorless. Then 
if more methyl green is added the neutral dye is slowly dissolved 
again; but as a rule neutral dyes are less soluble in excess of base 
than in excess of acid. 

As simple aqueous solutions of these dyes are impossible and as 
alcoholic solutions of dyes do not stain well, various methods are 
employed to secure their action on the tissues. In some instances 
they are kept dissolved by the presence of an excess of acid or base 
(particularly the former) ; in others a certain quantity of acetone or 
methylal is used to hold the neutral dye in solution ; sometimes (as 
in the original Romanovsky stain) the compound dye is used im- 
mediately after mixing, before the reaction is complete or pre- 
cipitation has taken place; or again (as in the Wright stain) methyl 
or ethyl alcohol may be used as a solvent, and then after applying 
the alcoholic solution to the slide it mav be diluted with water. 
This latter method is particularly efficacious, because the disso- 
ciation which takes place upon the addition of water causes the 
production of various dye compounds which may stain intensively 
and very selectively. 

87 



It is assumed that these compound dyes act on the protoplasm 
somewhat as follows : Certain parts of the cell have an affinity for 
the neutral stain and take it up as such; others, having an affinity 
for the basic dye, break up the neutral stain so as to obtain the 
basic portion of it, or if dissociation has taken place, take up the 
basic ion directly; while other parts of the cell with an affinity for 
acid dyes similarly combine with the acid portion of the stain. 
These three types of cell structures are known as neutrophile, 
basophile and oxyphile elements, respectively. The differentia- 
tion thus produced gives the neutral dyes their great value. 

Ehrlich's"Triacid Stain." 

The first neutral stain proposed for microscopic work was the 
"triacid stain" (seeEhrlich 1910, 1, 227). In forming this compound 
dye, acid fuchsin and orange G are mixed in solution and to the 
mixture is then added such a quantity of methyl green that there 
is still an excess of the acid dve. This excess of the acid dve allows 
the neutral stain to stav in solution. The dve thus formed is a 
very valuable blood stain, and brings out finely the different struc- 
tures in the leucocvtes. 

Slight modifications of this triacid stain have been used for 
tissues. The best known of these modifications is that of Biondi- 
Heidenhain. 

Eosin-Methylene-blue Compounds* 

The first worker to combine eosin and methylene blue was 
Romano vsky (1891). He realized that a mixture of these two dyes 
had great selective properties as a stain, and showed it to be ex- 
cellent for blood, particularly in bringing out the malarial parasite. 
He also appreciated that it was more than a mere mixture of the 
two dyes and that some new dye having the property of giving the 
nuclei a red color was present. It was some time later before the 
nature of this new dye was known, alt ho it was subsequently 
named azure I or methylene azure; its true chemistry has scarcely 
been understood until very recently (see p. 48). Methylene violet, 
which probably was also present, had already been described by 
Bernthsen (1885) . How these new dyes were formed in the Roman- 
ovsky stain was not known then; altho Romanovsky stated that 
different lots of methylene blue solution varied in their ability to 
give a good blood stain, and that old solutions on which a scum 
had formed were best. 

Present day blood stains are often spoken of as modified Roman- 
ovsky stains; altho the modifications are so great as to make them 
of a very different nature. The first modification was made by 
Nocht (1898) who concluded that the differential staining was due 



*A good account of the history of these blood stains is given by MacNeal (1906). 

88 



to the formation of other dyes by the decomposition of methylene 
blue. Unna (1891) had already described what he called poly- 
chrome methylene blue, made by heating a solution of methylene 
blue on a water bath with potassium carbonate. Nocht decided to 
use this in the Romanovsky stain instead of untreated methylene 
blue. He found that it gave very good results if properly neu- 
tralized before mixing with eosin; and then learned that better 
results could be obtained by the use of a smaller amount of alkali 
and a longer period of polychroming, without subsequent neutral- 
ization. 

The next step in preparing blood stains was made by Jenner 
(1899) who collected the precipitate formed when methylene blue 
and eosin are mixed, and redissolved it in methyl alcohol. He did 
not use polychrome methylene blue, and his stain lacked the 
nuclear staining principle of Romanovsky 's and Nocht's stains; 
but it was an important step in that he showed the possibility of 
collecting the precipitated compound stain and of dissolving it in 
some solvent other than water. Combining this procedure with 
the Nocht stain was the next logical step and was taken inde- 
pendently by Renter (1901) and by Leishman (1901). The method 
thus introduced was briefly to follow Nocht's technic of combining 
eosin with polychrome methylene blue, but then to filter off the 
precipitate and to redissolve it in methyl alcohol, not adding fur- 
ther water until the moment of applying the stain to the blood 
films. 

Modern blood stains are in general modifications of Leishman 's, 
differing only in detail. Wright's modifications, the one most used 
in America, (see Mallory and AYright, 1924, p. 170) differs from 
Leishman's only in that he prepared polychrome methylene blue 
by heating for only an hour in flowing steam, whereas the Leish- 
man technic calls for twelve hours at 65°, with subsequent stand- 
ing for ten days. Balch's modification calls for a polychrome 
methylene blue prepared by standing ten days with precipitated 
silver oxide. 

Giemsa's and MacNeal's modifications are somewhat different. 
Giemsa obtained methylene azure, in what he considered a pure 
form, and combined it with eosin in order to obtain a more definite 
compound than when polychrome methylene blue is used. Then 
to obtain better differentiation he mixed it with methylene blue. 
Following his instructions, the Griibler Co. put on the market a 
product known as Azure II, which was a mixture of Azure I (i.e., 
methylene azure) and methylene blue in equal parts; and also a 
compound known as Azure Il-eosin, which was an eosinate of 
Azure II, or more precisely a mixture of the eosinate of methylene 
blue with that of Azure I, in equal parts. This latter compound is 
the one generally known as the Giemsa stain. MacNeal (1922) 
proposed a method for obtaining a very similar blood stain, pre- 

89 



pared on even more scientific principles. This stain, known as the 
tetrachrome blood stain, is prepared by mixing definite proportions 
of methylene blue, methylene violet, methylene azure, and eosin. 
When first proposed (1922) this stain was to be prepared with a 
crude methylene azure, the pure product being at that time diffi- 
cult to prepare and therefore expensive. MacNeal's latest work, 
however (1925), shows a simple method of preparing methylene 
azure A (asymmetric di-methyl-thionin), which is apparently the 
important ingredient of Azure I, and he now specifies azure A in 
the tetrachrome stain instead of the less definite product methylene 
azure, as formerly. Azure A for this purpose can already be ob- 
tained on the market in America. 

Some difficulty was experienced at first in compounding the 
eosin-methylene-blue blood stains when imported dyes were no 
longer available. In some cases these difficulties were probably 
due to poor methylene blue or to poor eosin; but upon investiga- 
tion the solvent, rather than the dyes themselves, has been found 
to be most often at fault. As stated above, the precipitated com- 
pound dye must be dissolved in methyl alcohol; but there are many 
grades of methyl alcohol and not all are equally suitable for the 
purpose. Apparently absolute purity is not needed; but two points 
are very important; the methyl alcohol must be neutral in reac- 
tion, and it must be free from acetone. In specifying a methyl 
alcohol for the blood stains, these two properties should be in- 
sisted upon. Very good methyl alcohol for this special purpose is 
now on the market. 

Other Compound Stains 

Various other compounds of acid and basic dyes have been used 
for special purposes. The basic dye employed in these compounds 
is ordinarily methyl green or methylene blue; but sometimes basic 
fuchsin, pyronin or rhodamine is used. The most commonly used 
acid dyes are eosin, orange G and acid fuchsin; but certain others 
are occasionally employed. Picric acid forms a few useful com- 
pound dyes, rosanilin picrate (i.e., the compound of basic fuchsin 
and picric acid) being especially well known as a tissue stain. 

The Pappenheim panoptic triacid stain is a modification of 
Ehrlich's triacid compound. In this combination methylene blue 
or methylene azure is substituted for methyl green. It is a tissue 
stain of use in certain special technics. 

Ehrlich has proposed various other neutral stains, the best 
known being a compound of acid fuchsin and methylene blue used 
for staining blood; and a compound of narcein, an acid dye, with 
two basic dyes pyronin and methyl green or methylene blue. 



90 



CHAPTER IX 

THE NATURAL DYES 

AS STATED above (p. 11) the group of natural dyes is shrink- 
ing as more and more of them are being produced by artificial 
means. Alizarin, for example, used to be a natural dye of 
much importance; but now the artificial manufacture of this dye 
is much more economical. The group of natural dyes, as ordinarily 
recognized, contains only those which are not yet produced by 
artificial means. Indigo, however, is listed in this chapter, be- 
cause in its chemistry it does not fall in well with any of the groups 
of artificial dyes. Indigo is still obtained from the indigo plant, 
altho under present-day conditions its artificial manufacture is 
ordinarilv the more economical. 

The chemistry of the natural dyes is less definitely known than 
that of the artificial dyes. This is easily understood; for it will be 
recalled that there are two ways of obtaining information as to the 
chemistry of unknown compounds : the first by decomposing them 
into simpler compounds of known composition; and the second by 
manufacturing them from known compounds. In the case of dyes 
not yet prepared artificially the second of these two lines of pro- 
cedure is out of the question; hence the difficulty in learning their 
exact chemical structure. 

The most important natural dyes for the biologist are haema- 
toxylin, indigo, cochineal (and its derivatives), orcein, and litmus. 



The Indigo Group 

INDIGO c. I. NO. 1 17 7 

Synonym: Indigo blue. 

The plants from which indigo was formerly exclusively manu- 
factured are largely species of the genus known as Indigofera, altho 
some indigo-bearing plants are recognized by botanists as belong- 
ing to different genera. In these plants is a glucoside, indican, 
which is converted by fermentation into the dye indigo. Various 
formulae have been given for indigo; the one favored at present is 
based upon its method of artificial manufacture: 

CO CO 

\ / • 

c=c 

/ \ 

NH NH 

In this formula the exact chromophore group is uncertain; but the 
ketone group (CO) in a closed ring occurs so often in dyes that it is 
regarded as probably having chromophoric properties. 

91 





INDIGO-CARMIN C. I. NO. I180 

Synonym: Indigotine la. 
This is the sodium salt of indigodisulfonic acid : 

NaSOj CO CO SOjNa 

^rr \-/ ^rr 

\/\ / \ /\/ 

NH NH 

Indigo carmin is a blue dye of acid properties, which is sometimes 
used as a plasma stain in contrast to carmin, either mixed with it 
or following it.* 

Cochineal Products c. i. no. 1239 

Cochineal is a dye that has long been well known. It is obtained 
from a tropical insect generally known as the cochineal insect. By 
grinding and extracting the dried bodies of the female of the species 
in question a deep red dye is obtained, which is known as cochineal. 
By treating with alum this solution yields a product somewhat 
more free from extraneous matter, known as carmin. This is the 
form in which the dye is generalh^ obtained by the microscopist. 
Cochineal products are used in various ways in microscopic technic, 
generally as nuclear dyes. They are extremely valuable in cases 
where it is desirable to stain in bulk before sectioning. 

Cochineal, itself, has been used for various purposes in micro- 
scopic technic, even tho less used today than carmin. Alone it has 
little value, to be sure, for it has no direct affinity for tissues unless 
they contain iron, aluminium or some other metal. It is most 
commonly employed either with or following one of these mordants. 
A tincture of cochineal, that is an alcoholic solution containing 
calcium and aluminium chlorides, has been used by Mayer both on 
sections and for staining in bulk; but its most common method of 
use is with alum in watery solution. An alum-cochineal of this 
sort was first used independently by Mayer, Czokor, and Partsch; 
it can be used for sections, and is specially recommended for stain- 
ing in bulk, by which technic it stains nuclei violet red, and blood 
and muscle cells orange, while the cytoplasm is but weakly colored. 
A chrom-alum-cochineal has been used by Hansen for staining sec- 
tions. Spuler recommends an iron-alum-cochineal for staining in 
bulk when the sections are to be photographed, the technic bring- 
ing out nuclei, the blood in the tissues, and the muscle striations; 
sections may also be stained by the same method. By this technic 
the iron alum is applied first to the tissues as a mordant, and then 
followed by the stain. In Hansen's ferri-cochineal, on the other 

*For literature references to the procedures listed in this chapter see pp. 110 to 
128 and 138 to 1-io. 

92 



hand, the iron alum is mixed with the dye, and the mixture used for 
staining sections of tissue. 

Carmin is of considerable historic interest. It was used as early 
as 1839 by Ehrenberg, altho as we have seen (p. 7) not exactly for 
histological purposes. It was also employed in 1849 by Goppert 
and Cohn, by Corti in 1851, and by Hartig in 1854-8, these being 
the first uses of dyes in histology. It is still a valuable stain today, 
in spite of the enormous variety of synthetic dyes now available. 
On account of its freedom from toxicity it is useful for staining by 
injection. It is much used for staining in bulk, particularly in 
embryological work. A well known formula is Schneider's aceto- 
carmin, which is a valuable chromatin stain for fresh material in 
smear preparations. Alum carmin was used by Grenacher for 
similar purposes. Carmin is only slightly soluble in water at a 
neutral reaction; so solutions must be either acid (like the two 
above) or alkaline. Three alkaline formulae are of considerable 
use: ammonia carmin, which has been used both for injection and 
for staining sections; soda carmin, used primarily for injection; and 
Mayer's magnesia carmin, useful either for sections or for staining 
in bulk. Alcoholic solutions are also used: Grenacher's borax car- 
min (or as modified by Mayer) being a splendid nuclear stain for 
sections; and the hydrochloric carmin of Mayer serving both for 
sections and for staining in bulk. A special formula containing 
aluminium chloride (known as muci-carmin) has been proposed by 
Mayer and is used for staining mucin. In double staining it is 
sometimes used with indigo carmin; but most often with picric 
acid. Picro-carmin is a very well known combination used for 
double staining effects in sections, particularly for nervous tissue; 
it stains nuclei red and cytoplasm yellow. 

One of the most recent and important uses of carmin is in Best's 
carmin stain for glycogen. The method is simple and the result 
beautiful, the red glycogen standing out in sharp contrast to the 
blue of the nuclei after staining in alum haematoxylin. The stain 
is permanent; the method is of much importance both to the path- 
ologist and to the histologist. 

Carminic acid. The dye principle of carmin and cochineal is 
carminic acid. This product is obtained by extracting the insect 
bodies with boiling water, treating the extract with lead acetate or 
barium hydrate, and then decomposing the lead or barium carmin- 
ate with sulfuric acid. The exact composition of carminic acid is 
still somewhat uncertain; so far as known, it is: 

CH3 OH 

I CO I CeHiiOs 





HO / CO f OH 
COOH OH 

93 



It is a fairly strong dibasic acid and forms readily soluble salts with 
the alkali metals, and insoluble salts with the heavy metals. 
Aluminium carminate (obtained by precipitation from aluminium 
acetate and carminic acid or ammonium carminate) is soluble in 
aqueous or weak alcoholic solutions of acids. 

A slightly different aluminium compound, formed by mixing 
alum and carminic acid is used in histology. This combination was 
called carmalum by Mayer, and has also been used by Grenacher 
and Rawitz; it is a useful nuclear stain for sections, and is often 
employed with light green or indigo carmin as a contrast stain. A 
so-called muci-carmin, an acid solution containing aluminium 
chloride, has beem employed by Rawitz to stain mucin; while 
Mayer's para-carmine, containing aluminium and calcium chlo- 
rides, is used both for sections and for staining in bulk. By others 
a combination of iron with carminic acid has been used for similar 
purposes. 

Carmein. Carmin, kept in ammoniacal solution, changes in its 
properties, due to oxidation. The oxidized carmin, often known as 
carmein, can be obtained by treating a carmin solution with hy- 
drogen peroxide and precipitating with alcohol. It is a dark colored 
mass which can be ground into a black powder. 

Orcein and Litmus c. i. no. 1242 

Both orcein and litmus are obtained from certain lichens, Lecan- 
ora tinctoria and Rocella tinctoria. These lichens are colorless, but 
when treated with ammonia and exposed to the air, blue or violet 
colors develop. The colors are due to certain acids, one of which is 
orcin : 

OH 

/ 

\ 
OH 

Orcin, acted upon by air and ammonia, becomes orcein. 

ORCEIN 

The exact formula of orcein is unknown. It is a weak acid, 
soluble in alkalies, with a violet color. 

In alcoholic solution Unna has used orcein for staining elastin 
tissue; he has employed it for connective tissue, following poly- 
chrome methylene blue; and for plasma fibrils in the epithelium, 
following anilin blue; also with anilin blue or acid fuchsin in study- 
ing the process known by him as chromolysis. It has found less 
frequent use among other histologists; but has been employed by 

94 



Israel in acetic acid solution for staining sections (nuclei staining 
blue, cytoplasm red); and by Moll, dissolved in weak hydro- 
chloric acid, for staining sections of embryos. 

LITMUS 

The exact composition of litmus is likewise unknown. It is ob- 
tained from the same lichens as orcein, treating them with lime 
and potash or soda, in addition to air and ammonia. Its colored 
principle is known as azolitmin. 

Litmus scaj-cely needs comment here. It is a feeble dye and is 
never used as an histological stain. Its classic use is for indicator 
purposes; but it is now coming to be largely replaced by the various 
synthetic dyes (especially sulphonphthaleins) which change color 
thru an hydrogen-ion range near the neutral point. 

Brazilin and Haematoxylin 

The two natural dyes, haematoxylin and brazilin are closely 
related chemically and upon decomposition yield the two com- 
pounds, pyrocatechin 

OH 




and pyrogallic acid 

OH 



OH 
OH 

OH 

Both dves are obtained bv extraction of the bark of certain trees, 
haematoxylin from logwood and brazilin from brazil wood (red 
wood). Both trees are legumes and belong to the family Cesal- 
piniaceae; they are found only in the tropics. Haematoxylin 
comes from a single species; while brazil wood is a term applied to 
various different species all yielding brazilin. 

BRAZILIN C. I. NO. I243 

The composition of this substance is supposed to be: 

HO /\ O CH, 



_ C— OH 

CH \ 
I CH. 

/ \ 

\ / 

I I 
HO OH 

95 



Its solution is colorless, but it becomes red on exposure to tlje air, 
as it is then oxidized into the dye brazilein, which probably has the 
formula : 

HO 




HO O 



With alum it is used as a nuclear stain (known as brazalum) by 
Mayer. It is also used by Hickson for similar purposes following 
treatment with iron alum as a mordant. 



HAEMATOXYLIN 



C. I. NO. 1246 



Haematoxylin is a homolog of brazilin, having one more hy- 
droxyl group, the generally accepted formula being: 



OH 



HO 



O CH 



CH 



C— OH 
CH. 



<z> 



HO HO 

Like brazilin it is not a dye, but its color develops in solution upon 
standing, due to the oxidation into haematein, which is homologous 
to brazilein and probably has the formula: 

OH 
HO_/\_0_CH. 



C— OH 
CH. 



V 



HO O 



Haematoxylin is without question one of the most important 
biological stains. It is as valuable to the cytologist and histologist 
as methylene blue is to the bacteriologist; and probably is second 



96 



only to methylene blue in the number of different purposes for which 
it is used. It is valuable not only because it is a powerful nuclear 
stain and a chromatin stain par excellence, but also because it has 
striking polychrome properties. With the proper differentiation it 
is possible to get several shades intermediate between blue and red 
to show in the same preparation. 

Haematoxvlin is seldom used alone, as it has little affinitv for the 
tissues in itself, even after "ripening" when it is largely converted 
into haematein. Some form of mordanting is ordinarily required; 
and most of the haematoxylin formulae either call for some metallic 
salt or specify previous treatment of the sections with one. In 
plant histology, however, there is some use for haematoxylin alone. 
Its greater affinity for plant than for animal tissue implies the 
presence of aluminium, copper, or iron in the former. In fact 
haematoxylin can be used as a very delicate reagent for iron or 
copper. 

Perhaps the best known formulae for staining with haematoxy- 
lin are the combinations with aluminium, generally in the form of 
alum. Bohmer's alum haematoxylin (1865), altho no longer used, 
is of much historic interest as it was the first stain of this type to be 
used. The best known at present is Delafield's alum haematoxy- 
lin, which is very useful tissue stain with great affinity for chroma- 
tin and nuclei, and has much value in staining cellulose walls in 
vascular plants. Another alum haematoxylin used for similar 
purposes is that of Ehrlich. 

Mayer's haemalum is another well known alum combination. 
In this stain haematein is first prepared and then combined with 
alum. The name haemalum, proposed by Mayer, is now generally 
accepted for this combination, and various other haemalum form- 
ulae have since been proposed. They are useful chromatin stains 
and are called for in various special procedures. 

Mayer has also combined haematein with aluminium chloride, 
his haemacalcium calling for this salt and calcium chloride, while 
his muc-haematin contains aluminium chloride and glycerin. The 
latter is used for staining mucin. 

The iron combinations are perhaps equally valuable. The 
original iron haematoxylin was that of Benda; but the best known 
at present is M. Heidenhain's, which is one of the most useful 
histological and cytological stains, both in botany and zoology. It 
is a powerful stain for chromosomes and centrosomes, and is of use 
for bringing out the middle lamellae in wood. Various other 
modifications of iron haematoxvlin have been used, but thev are 
all similar in principle. Ordinarily the iron salt is not mixed with 
the stain, but is used for a preliminary mordanting of the tissue. 

Haematoxylin has been combined with chromium, one of the 
early staining methods being that of R. Heidenhain, which called 
for potassium bichromate as a mordant. Various recent modifi- 
cations are in use today, such as that of Apathy, for staining general 

97 



tissue. Weigert uses a chrom combination for staining nervous 
tissue. 

Benda uses haematoxylin following treatment with a copper salt 
for studying spermatogenesis; and Bensley a similar technic for 
chromosomes and mitochondria. 

Mallory has proposed a formula for haematoxylin containing 
phosphomolybdic acid and also one containing phosphotungstic 
acid. The latter method is especially valuable for staining cells in 
the process of mitosis, and for distinguishing fibroglia, myoglia and 
neuroglia fibrils from collagen and elastin fibrils, especially in 
tumors, but also in normal tissues. It brings out sharply the 
striations in skeletal and cardiac muscle fibers. Haematoxylin is 
used in combination with other stains, especially eosin, but not so 
frequently as in the case of the common anilin dyes. The Van 
Gieson technic calls for haematoxylin followed by picric acid and 
acid fuchsin. A few other methods call for picric acid or ammonium 
picrate after haematoxylin; and it is sometimes used with eosin or 
after orange G or acid fuchsin. Most of these combinations, how- 
ever, are called for only in the case of special procedures. 

At present it is possible to obtain very satisfactory haematoxy- 
lin manufactured in America. A very good statement of the situa- 
tion is given by McClung (1923) . The first American haematoxylin 
put on the market, altho pronounced very good by many of those 
using it, still proved to be lacking in some of the qualities which the 
best haematoxylin ought to possess. Thru the cooperation of the 
manufacturers, the trouble was located. In order to obtain a pure 
product, SO2 had been used in the manifacture for bleaching pur- 
poses; but this treatment was found to be harmful to the haema- 
toxylin. The product now on the market is not so treated, how- 
ever, but an extra recrystalization is introduced in the place of the 
bleaching process; and the resulting product meets all the tests to 
which it is submitted. 



98 



CHAPTER X 

THE THEORY OF STAINING 

THRUOL T the preceding pages of this book an effort has been 
made so far as possible to avoid theoretical discussions. Altho 
thev contain some statements the truth of which cannot be 
regarded as fully established, as in the case of the chemical com- 
position of some of the dyes, the discussion in general has been con- 
fined to observations and to chemical information for which there 
is good authority, without any attempt to introduce explanations 
of a theoretical nature. The present chapter, therefore, was not 
part of the original plan of the book, and the decision has finally 
been made to introduce it merelv because it is felt that a brief 
statement of some of the most probable theories to explain staining 
may be of value in assisting the histologist in the intelligent use of 
stains for his purposes. 

A long theoretical discussion of this subject might be included 
here, basing it upon the lengthy arguments supporting the various 
theories that have appeared in the literature. Such a detailed dis- 
cussion, however, would probably be of little value. Hence this 
chapter is confined to a bare outline of the important points of the 
different theories. 

Theoretically the dyeing of textile fabrics and the staining of 
microscopic structures are the same. In one case only the gross 
effects are observed, in the other the microscopic details. Any 
theory, therefore, that will explain the details of microscopic stain- 
ing will be fully adequate to account for dyeing in bulk. 

Theories to account for dyeing or staining have in general been 
based exclusively upon either physical or chemical phenomena. It 
would seem at first thought that the dves combine so firmlv with 
the tissues stained by them that the phenomenon must be a 
chemical one; but the exponents of physical theories have taken 
pains to show that all the observed facts can be explained on a 
physical basis, and that some observations are hard to explain if a 
chemical union between tissue and dye actually takes place. In a 
chemical union a new substance is formed which does not neces- 
sarily have the properties of either substance entering into its 
formation, and it is ordinarily impossible to recover the original 
substances by means of simple solvents, ^^hen tissue is stained 
there is no evidence of any new substance having been formed, the 
colored tissue merely taking on one of the characteristics of the 
dye (color) in addition to the properties which it originally possess- 
ed; it is, moreover, ordinarily possible to extract all or nearly all of 
the color by sufficiently long immersion in water, or by the fairly 
brief action of alcohol. Another observation which points against 

99 



chemical action is that the tissue never removes the dye completely 
from solution, even tho very dilute; whereas ordinary chemical 
reactions tend to continue until one of the components of the re- 
action is exhausted. Such facts as these, to the exponents of the 
physical theory, are enough to refute the possibility of chemical 
action. 

It has, indeed, been pointed out that all of the ordinary dyeing 
or staining phenomena can be explained on a physical basis. It is 
evident, to be sure, that the action of the dyes is not confined to the 
surface of the material colored; but as the substances stained are 
always more or less porous, absorption of the dye after passing thru 
cell membranes by osmotic action accounts for the penetration. To 
account for the selective action of different dyes upon different 
parts of the cell it is possible to use the principle of absorption as an 
explanation. Adsorption is the property possessed by a solid body 
of attracting to itself by purely physical means from a surrounding 
solution certain compounds or ions present in that solution. Hav- 
ing once entered the tissue it is possible that the dyes remain there 
in a state of solid solution, similar to that in which gold is retained 
in ruby glass. This possibility, in the opinion of those who hold the 
physical theory, is the more likely because a dye causes the tissue 
to become the same color as the dye shows in solution, but not 
necessarily the same as it shows in its solid form. Dry fuchsin, for 
example, is green; its solution, however, is red, and so are tissues 
stained by it, no matter how completely they may be dried. 

Some of those who hold in general to the physical theory of stain- 
ing admit that these simple physical phenomena alone cannot ex- 
plain everything, as for example, instances in which a dye pene- 
trates different cell elements equally readily, but can be easily ex- 
tracted from some of them while scarcely at all from others. It is 
assumed, therefore, that the dyes penetrate the cells by mere ab- 
sorption and diffusion, but are in some cases precipitated there by 
acids or bases, or other chemical reagents present, thus preventing 
their extraction by simple solvents. Such a theory admits the 
possibility of chemical action without assuming an actual chemical 
union between the dye and the tissue. 

In this connection the action of mordants is interesting. Some 
tissues do not stain directly with certain dyes, or if they do the 
color is very feeble. If, however, they are treated previously or 
simultaneously with certain chemicals, the dyes "take." 

It is possible that this phenomenon may be due to chemical 
affinities of the mordant for the tissue on the one hand and the dye 
on the other; but the special value of iron and aluminium salts as 
mordants makes it seem quite probable that their action may be 
actually to precipitate the dye in the tissue. To the bacteriologist 
the behavior of different bacteria to the Gram stain immediately 
suggests itself. In this technic (see p. 68) one of the methyl violet 
dyes is allowed to act on the bacteria for a definite length of time, 

100 



and is then followed by treatment with iodine. After that, alcohol 
is applied for decolorization; but it proves that certain kinds of 
bacteria retain the violet stain even after counterstaining with some 
dye of a different color; while others are readily decolorized by the 
alcohol, and take the counterstain. According to some, this action 
is accounted for by assuming that the iodine combines with the 
methyl violet inside the cell, converting it into a molecule so large 
as to be unable to pass thru the cell membrane again. It may, on 
the other hand, be assumed that there is an actual chemical differ- 
ence between Gram-positive and Gram-negative bacteria, so that 
the former combine chemically with the iodine and dye, while the 
latter do not. Neither theory has been definitely proved or dis- 
proved. 

Evidence is still lacking, in fact, to prove or disprove either the 
chemical or the physical theory as it relates to general staining. The 
difference, perhaps, is not one of immense importance. It is fre- 
quently pointed out that there is no sharp distinction between 
chemistry and physics, and in such delicate reactions as those in- 
volved in staining, we may be well in the borderland between the 
two branches of science, where it is impossible to say that a given 
phenomenon is purely physical or purely chemical. There are, 
however, certain chemical principles distinctly different from the 
physical ones just mentioned, that may well enter into the phe- 
nomenon of staining; and it is these that are considered most im- 
portant by the exponents of the chemical theory. 

It is pointed out on behalf of the chemical theory that just be- 
cause physical forces alone can explain the facts, one is not justified 
in assuming that chemical unions do not take place when the 
opportunity for them is present. It is kno^m that some parts of 
the cell are acid in reaction, others alkaline; and it is a well kno^m 
chemical principle that the former would tend to combine with the 
kations in solutions with which they come in contact, the latter 
with the anions. Now inasmuch as in certain dyes the color exists 
in the kation (basic dyes) and in others in the anion (acid dyes), it 
is natural to expect chemical combinations to take place between 
dye^and tissue, depending upon the reaction of the latter. Argu- 
ments for the physical theory which exclude chemical action must 
furnish strong proof that no chemical union occurs; and those who 
favor the chemical theory claim that such proof is lacking. That 
the stained tissue does not present any characteristics to the eye 
not possessed by either tissue or dye before staining does not prove 
that no new substance has been formed, nor is this claim proved 
by the fact that sufficiently long action of solvents removes the 
color. Alcohol and even water are not absolutely inert chemically 
and may withdraw the dye by chemical instead of physical action ; 
the very length of time necessary to remove the color completely 
(sometimes so long as to allow bacterial decomposition of the tissue) 
indicates that a rather strong union between dye and tissue has 

101 



taken place. As to the fact that a tissue which has a strong affinity 
for some particular dye never withdraws that dye completely from 
a very dilute solution, those who favor the chemical theory point 
out instances where chemical reactions are known to take place 
and yet to stop before either component is exhausted; and they 
further claim that chemical action is strongly indicated by the fact 
that in dilute solutions the tissues take up relatively larger quan- 
tities of the dye than in concentrated solutions. 

In brief, the chemical theory of staining is that the tissues have 
certain definite chemical affinities which are satisfied by the chem- 
ical affinities of the dyes; tnerefore, when the tissue is put in a 
solution of the dye the latter combines with those portions of the 
tissue or of the individual cells which have the proper chemical 
nature. This theory, it will be seen, is especially well adapted to 
explain the differential staining which takes place, as when we find 
a certain stain acting only on tde nuclei or even exclusively upon 
certain structures within the nuclei. The chemical theory is not 
yet firmly established, and the probabilities are that staining is 
both a chemical and physical phenomenon; but it is coming to be 
more widely accepted than the physical theory. Hence it deserves 
a more detailed discussion. 

The chemical theory of staining is dependent largely upon the 
question of the acid or the basic character of the dye molecule. It 
will be recalled that all ordinary dyes are encountered either as 
sodium or potassium salts of dye acids or as dye salts of colorless 
acids, the former being the acid dyes and the latter the basic dyes; 
while certain compound stains are neither acid nor basic dyes, in- 
asmuch as the property of color exists in both the anion and the 
kation. 

Briefly stated, the fundamental chemical theory of staining is 
that certain parts of animal or plant cells are acid in character and 
hence have an affinity for the basic dyes. The nuclei of the cells, 
or especially the chromatin within the nuclei, are assumed by this 
theory to be acid in character (due largely to their constituent 
nucleic acid) , and there is no question but that they have a strong 
affinity for basic dyes; while the cytoplasm has an affinity for acid 
dves and is assumed to be basic in character. 

Now this is by no means the whole of the chemical theory of 
staining. The theory is so complex and has so many ramifications 
and special applications that it takes an intensive study of the 
subject to comprehend it fully. It is well knowni for instance, that 
certain basic dyes have stronger affinities for certain parts of the 
nuclei than for others, and that of the various cytoplasmic struc- 
tures outside of the nucleus some are more readily stained by 
certain acid dyes and some by others. Such special applications 
as these, of course, are not explained on the theory of acid or basic 
character alone. It is possible, for example, to use the Flemming 
triple stain, which employs the acid dye orange G and the two basic 

102 



dyes safranin and gentian violet, thus staining the chromatin with 
gentian violet and the rest of the nucleus with safranin. It is 
difficult to say just how any chemical theory of staining can yet 
satisfactorily explain such selective action as this. It is, indeed, 
admitted by some upholders of the chemical theory that the chem- 
ical action of dyes is not specific, and merely serves to differentiate 
acid from basic elements of the tissue; and that the further differ- 
entiation, as between chromatin and other parts of the nucleus, is 
due to physical forces, thus indicating a difference in the structure 
rather than in the chemistry of the different portions of the nucleus. 
The neutral stains have a very interesting action. When tissue 
is stained with them they seem to break up partlj^ into their com- 
ponent acid and basic elements and stain portions of the tissue as 
the simple dyes would individually. But in addition the neutral 
stain itself seems to have an affinity for certain parts of the tissue 
and hence a third color is possible. This explains in part the poly- 
chrome effects obtained by the eosin-methylene-blue blood stains — 
only partly so, however, because in preparing these stains methy- 
lene blue breaks up into certain other dyes so that more shades than 
those expected from eosin and methylene blue alone are obtained. 
So far as the action of stains is chemical, their use forms a con- 
necting link between the two subjects, histology and micro- 
chemistry. These two branches of science are generally thought 
to be entirely distinct. The histologist, with the technic and view- 
point of the biologist, prepares delicate sections of various materials 
colored with one or more of a long series of available dyes, and 
studies the biological structures present under the microscope. The 
microchemist, with the technic and vie^\'point of the chemist, 
examines with the microscope similar material treated with various 
reagents of known chemical reaction, and from his observations 
draws conclusions as to the chemical nature of the substances 
examined. There is some possibility, however, that the difference 
between histology and microchemistry is one of point of view rather 
than of methods. Both the microchemist and the histologist 
study the action of chemical compounds on substances or struc- 
tures visible under the microscope; the difference is that the micro- 
chemist uses the chemical compounds in question as chemical re- 
agents while the histologist uses his as dyes to color the microscopic 
structures and thus to increase their visibilitv. 

Now, on the chemical theory of staining, the biologist is using 
complicated chemical reactions in his microscopic technic — so 
complicated in fact as to be unintelligible chemically. To bridge 
the gap between histology and microchemistry these reactions 
must be made intelligible. The first step in this direction has now 
been taken by Unna (1921) in a very important contribution to 
the subject of cell chemistry. He points out the need of harmoniz- 
ing chemical and histological investigations and proposes a method 
of doing this which he calls chromolysis. The technic he has de- 

103 



veloped, altho quite complicated in its details, can be summed up 
briefly as follows : To select a dye, or a mixture of dyes, either acid 
or basic, which bring out some intracellular structure whose chem- 
istry it is desired to learn; then to submit the sections to the action 
of various solvents, beginning with simple cold water, next pro- 
ceeding to hot water, and from that to the more powerful solvents, 
but using only those whose action on proteins, lipoids or carbo- 
hydrates is known to the chemist; then to stain the sections with 
the staining fluid selected; and finally to determine by microscopic 
examination which solvents have removed the substance under 
investigation. 

By such methods as these Unna hopes to make considerable 
progress in the microchemistry of the cell; and it will be readily 
seen that once the gap between chemistry and histology is bridged, 
progress will become constantly more and more rapid. As soon as 
it is possible to obtain reasonable hj'^potheses as to the chemical 
nature of the various intracellular bodies in any one particular 
type of tissue, then it will be possible to ascertain the affinities of 
the different dyes now used in histology for the different chemical 
compounds thus recognized; and then by using the same stains on 
other tissue it will be possible to apply the information thus ob- 
tained to the solution of the chemistry of other microscopic struc- 
tures. In other words, stains will become chemical reagents in- 
stead of merely dyes for making microscopic structures visible. In 
this way it is hoped that chemistry and histology, working to- 
gether, may solve some of the obscure problems as to the nature of 
the cell and its contents. 



.». -'•*'■" 



.1'/ 

i 



104 



APPENDIX I 
TABLES RELATING TO STAINS 

In the following tables all the dyes that are frequently mentioned in the literature 
dealing with microscopic technic are listed, together with the most important uses 
of each in the biological laboratory. The list of uses (Table 2) is necessarily 
incomplete. In the case of the most commonly used stains, in particular, it 
has been necessary to group various uses together under some general term, 
without attempt to list the individual procedures. In general, however, the policy 
has been to list the methods for which a stain is most commonly used today, and 
of the obsolete methods to give only those of historical interest. Criticism will 
be welcomed from anyone noticing any serious omission. 

The dyes in Tables 1 and 2 are listed in the same order as in the main part of 
this book. Hence either the general Table of Contents or the list in Table 1 may 
be used to learn the order of the stains when it is desired to find some particular 
one in Table 2. For an alphabetical list referring to the following pages see the 
general Index, 

The references given in the last column of Table 2 refer ordinarily by name and 
date to the literature listed in Appendix III. Five references, however, are used 
so often that the date is omitted: they are referred to merely as Chamberlain, Lee, 
Ehrl. I, Ehrl. II, and Mai. & Wr. These refer respectively to: Chamberlain's 
Methods in Plant Histology (1924), Lee's Microtomists Vade-mecum (1921), 
Ehrlich's Enzykopadie der Mikroskopischen Technic (1910), Vol. I and Vol. II, 
and Mallory and Wright's Pathological Technic (1924). Xo effort has been made 
to give the original reference in all cases ; but rather to refer to some readily available 
description of each technic than can be followed by anyone using the procedure. 






105 




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128 



Table 3. A List of Biological Stains Grouped According to the Field in 

Which Used.* 

animal histology 
Nuclear stains (basic) 

Janus green B. 

Thionin 

Methylene blue 

Toluidin blue 

Cresyl violet 

Safranin 

Magdala red 

Auramin 

Fuchsin 

Hoffman's violet 

Iodine green 

Gentian violet (including'crystai and methyl violets) 

Cochineal and carmin 

Orcein 

Brazilin 

Haematoxylm 

Cytoplasm stains (acid) 

Picric acid 
Orange G. 

Bordeaux red 

Fast yellow (bone tissue) 

Methyl orange (for keratin in skin) 

Amaranth (nervous tissue) 

Biebrich scarlet W. S. 

Bismarck brown 

Congo red 

Tr^'pan red (vital staining) 

Benzopurpin -iB 

Tr\T)an blue (vital staining) 

Alizarin red S (for nervous tissue) 

Neutral red (largely for vital staining) 

Nigrosin 

Malachite green 

Light green SF yellowish 

Acid fuchsin 

Methyl blue 

Anilin blue W. S. 

Rhodamine 

Eosin Y. 

Eosin, ale. sol. 

Erythrosin (nervous tissue) 

Indigo carmin 

Fat stains 

Sudan III 
Sudan IV 

Nile blue sulfate. 



*In this table the stains in bold faced type are those of widest application. 
When a stain is specified for practically only one purpose that purpose is mentioned 
in parenthesis; the stains not so designated are of fairly general application in the 
particular field under which they are listed. 

U9 



PLANT HISTOLOGY 

For lignified cell walls 

Methylene green 

Safranin 

Iodine green 

Gentian violet (including crystal and methyl violets) 

Methyl green 

For cellulose walls 

Congo red (plant mucin) 

Malachite green (for host tissue in case of fungus diseases) 

Light green SF yellowish (cellulose walls) 

Acid fuchsin 

Eosin Y 

Erythrosin. 

Haematoxylin 



CYTOLOGY 

General nuclear stains (basic) 

Thionin 

Methylene blue 

Toluidin blue 

Magdala red 

Fuchsin 

Gentian violet (including crystal and methyl violets) 

Methyl green 

Carmin 

Orcein 

Haematoxvlin 



Special chromatin stains 

Alizarin red S 

Thionin 

Methyl green 

Safranin 

Gentian violet (including crystal and methyl violets) 

Iodine green 

Carmin (foi fresh cells) 

Haematoxvlin 



Cytoplasm stains (acid) 

Picric acid 
Orange G. 

Bordeaux red 
Methyl orange 
Acid fuchsin 
Eosin Y. 

Stains for mitochondria, etc. 

Aurantia 
Janus green B 
Acid fuchsin 
Crystal violet. 

130 



PATHOLOGY AND BACTERIOLOGY 



Nuclear stains (basic) 



Thionin 
Methylene blue 

Toluidin blue 

Safranin 

Cresyl violet 

Fuchsin 

Hoffman's violet 

Iodine green 

Gentian violet (including crystal 

and methyl violets) 
Pyronin 

Cochineal and carmin 
Orcein 
Haematoxylin 



Blood stain constituents 

Orange G. (acid) 
Narcein (acid) 
Methylene blue (basic) 
Methylene azure (basic) 
Neutral red (acid) 
Acid fuchsin (acid) 
Methyl green (basic) 
Pyronin (basic) 
Rhodamine (acid) 
Eosin Y (acid) 

Fat stains 

Sudan IV 

Nile blue sulfate. 

Bacterial stains 



Cytoplasm stains (acid) 



Picric acid 

Martins yellow (for cancer 

tissue) 
Orange G. 
Methyl orange 

Amaranth (for nervous tissue) 
Biebrich scarlet W. S. 
Congo red 
Alizarin red S. 
Neutral red 
Nigrosin 

Malachite green Used 

Light green SF yellowish 
Acid fuchsin 
Methyl blue 
Anilin blue, W. S. 
Eosin Y. 
Ervthrosin 



Bismarck brown (Gram counter- 
stain) 

Thionin 

Methylene blue 

Safranin (Gram counterstain) 

Fuchsin 

Gentian violet (including crystal 
and methyl violets) 

Methyl green (constituent of 
Pappenheim stain) 

Pyronin 

Erythrosin 

Rose bengal 

in bacteriological media 

Neutral red 
Fuchsin 
Acid fuchsin 
Brilliant green 

Methylene blue 
Eosin Y. 



131 



APPENDIX II 
COMMISSION SPECIFICATIONS OF CERTAIN STAINS 

As announced in several notes published by the Commission in Science, it is 
planned to draw up specifications for all the stains put on a certification basis. 
So far as these specifications have been prepared they are given on the following 
pages. Similar specifications for other stains will be prepared later. 

It must be understood that these specifications are not intended to furnish 
definite statements as to the chemistry of satisfactory stains. The object with 
which they were drawn up was to allow the manufacturers as much latitude in the 
matter of chemical composition as has been found consistent with good results in 
practice, and to lay the greatest stress upon the performance of the stains in actual 
laboratory use. The requirements listed are those which must be met by dyes 
submitted to the Commission for certification. 

These specifications are published, moreover, with the understanding that they 
are subject to revision at any time. Further investigations are in progress con- 
cerning the adaptability of various dye products for different purposes, and also 
as to the relation of optical characters to performance in staining. The Commission 
reserves the right to modify the specifications for any stain in regard to either of 
these two points as data accumulate to show the need of modification. 

SPECIFICATIOXS FOR METHYLENE BLUE 

1. Samples of methylene blue to be considered must be of the so-called medicinal 
grade. It is expected that they will meet the U. S. P. requirements, but less weight 
will be attached to this consideration than to those following. In other words, a 
sample giving satisfactory performance will not be excluded because of failure in 
some particular to meet these chemical requirements. 

2. Methylene blue for the purpose above specified must contain at least 75 
per cent total color, this to be determined by reduction with titanous chloride. 
When reduced by titanous chloride in an atmosphere of carbon dioxide, 1 gram of 
the dye must consume at least 4.69 cc. normal titanous chloride solution. 

3. The methylene blue must have no solvent action on casein. This is to be 
determined as follows: Prepare two 1 per cent solutions of this stain, one in distilled 
water, the other in tap water. Place single drops of skimmed milk on each of two 
clean glass slides and smear each drop over a surface of about one square centimeter 
so as to form a very thin film of milk; allow this film to dry without heat or at a 
temperature not over 60°C., immerse for about a minute in xyol to dissolve the fat, 
then for the same length of time in alcohol to coagulate the casein. After this 
immerse one slide in the distilled water solution of methylene blue and the other 
slide in the tap water solution, allowing them to stand for three minutes; at the 
end of this period there should be no action of the stain on the casein. 

4. The methylene blue should stain the diphtheria organism in any of the types 
of solution^ ordinarily employed. It should be tested as follows: Prepare three 
solutions of the stain, one a 1 per cent solution in distilled water, the second a 
mixture of three parts saturated alcoholic solution to 10 parts of distilled water, 

1S2 



and the third three parts of saturated alcoholic solution to 10 parts of 0.01 per cent 
NaOH. Prepare three slides of a fresh culture of a diphtheria organism; stain one 
slide in each of these three solutions for two or three seconds only, i. e., just as 
briefly as the stain can be poured on and poured off, and wash each slide im- 
raediatly. Examined under the microscope all three of these preparations should 
show deeply stained bacteria with the characteristic metachromatic granules 
suflSciently distinct to insure accurate diagnosis. 

5. The sample should prove satisfactory for histological use. Xo exact method 
for determining this can be given, but the sample must be submitted to one or two 
experts in histological technic in order to get their judgment. 

6. It must be understood that these standards refer to samples to be used for 
ordinary bacteriological and histological staining. Special standards for methylene 
blue used in vital staining will undoubtedly be necessary. These standards, how- 
ever, have not yet been determined. 

SPECIFICATIONS FOR SAFRANIN' O 

1. Samples of safranin O must be of the t^-pe represented by Colour Index 
No. 841 and on spectrometric analysis .should have an absorption curve maximum 
at approximately olou/x as determined in a one cm. layer by a spectrophotometer. 
Other dyes must not be present. 

2. Safranin samples to be certified by the Commission must contain at least 75 
f>er cent total color as determined when reduced V>y titanous chloride in an atmos- 
phere of carbon dioxide. One gram of the dye must consume at least 4.195 cc. 
normal titanous chloride solution. 

3. The samples should prove satisfactory for histological use. No exact method 
for determining this can be given, but the samples must be submitted to one or two 
experts in histological technic in order to get their judgment. Their judgment 
must be based to a considerable extent upon the behavior of the stain in the Flem- 
ming triple staining technic, in which it is used together with orange G and gentian 
violet. In other words, the stain must be of such a shade as to contrast well with 
both of these two other dyes. 

SPECIFICATIONS FOR BASIC FUCHSIN 

1. Basic fuchsin designed for staining and indicator purpo.ses must be ro.saniliu 
or new fuchsin (Colour Index No. 678) or else a mixture of rosanilin and para- 
rosanilin containing at least half of the former (that is, c-orresponding to Colour 
Index No. 677). 

2. Fuchsin samples to be certified by the Commission must be of such a strength 
that, when reduced by titanous chloride in an atmosphere of car])on dioxide, one 
gram of the dye will consume at least 46.5 cc. normal titanous chloride solution. 
A sample of this strength will be between 76 and 85 per cent total dye content, the 
exact dye content varying according to the relative amounts of the higher and the 
lower homologs present. 

8. The sample should prove satisfactory for staining the tuhercle organism and 
should retain its color sufficiently when treated by the Ziehl metliod t» be diagnostic 
when staining tubercular discharges. This must be determined by an investigator 
skilled in this particular technic. 

133 



4. The sample must prove satisfactory for use in the Endo medium. In making 
this test the following technic should be used: A saturated alcoholic solution is 
diluted 2 to 3 times, the dilution to be such that no precipitation occurs when mixed 
with a sodium sulphite solution. Then add 0.5 cc. of the saturated and the diluted 
fuchsin solutions each to 10 cc. of a 2.5 per cent sodium sulphite solution. Select 
the strongest of these which shows no precipitate and add it to the other ingredients 
of Endo agar, sterilize and cool. It should then be colorless, but the color must be 
restored by the colon and dysentery organisms when inoculated upon it. The test 
must be made by one familiar with the technic in question. 

5. It must be understood that as basic fuchsin is used in other special forms of 
technic, new standards may be called for. The present specifications apply par- 
ticularly to the above mentioned two uses: but samples fulfilling them are ordi- 
narily satisfactory for all histological purposes. 

SPECIFICATIONS FOR ACID FUCHSIN 

1. Acid fuchsin designed for staining and indicator purposes must correspond 
to Colour Index No. 692. Inasmuch as the dyes of this type on the market are 
mixtures of greatly varying composition, and it has not yet been shown that any 
one of the components of these mixtures is better for biological purposes than any 
of the others, no definite physico-chemical specifications are made at the present 
time, with the reservation that more specific requirements may be drawn up at a 
later date if future work shows them to be desirable. 

2. Acid fuchsin samples to be certified by the Commission should be of such a 
strength as to consume not less than 2.0 cubic centimeters of normal TiCls solution 
per gram of sample. Such samples would have a total dye content of approximately 
60 per cent. 

3. Acid fuchsin samples for certification should prove satisfactory counter- 
stains in general histological work. They should in particular prove satisfactory 
in the Bensley technic for staining mitochondria, and in the Mallory connective 
tissue stain (with anilin blue and orange G). They should be tested for these 
purposes by histologists skilled in the technic in question. 

4. The samples shall prove satisfactory for use in the Andrade indicator. For 
this purpose they shall be tested as follows: Dissolve 0.2 gram in 100 cc. water, 
neutralize by adding between 15 and 25 cc. of Normal NaOH. When neutralized 
with the proper amount of the reagent, the solution should become a straw color 
upon standing and should impart no noticeable color to ordinary bacteriological 
nutrient agar when added at the rate of 1 per cent. The red color should be 
restored in this medium by the colon organism when growing in the presence of 
any sugar attacked by it. 

5. Labels to be used on the acid fuchsin samples should state the formula giving 
best results with the sample in question in preparing the Andrade indicator. 

SPECIFICATIONS FOR GENTIAN AND CRYSTAL \^OLET 

1. Crystal violet samples shall contain no dye except hexa-meth\l pararosanilin 
(Colour Index No. 681), having its absorption maximum at 590fifi when de- 
termined in a cell of 1 milimeter thickness in a spectrophotometer. 

134 



i. Crystal violet samples to be certified by the Commission must be of at least 
80 per t!ent dye content as determined by reduction of TiCls in an atmosphere of 
carbon dioxide. 

3. Gentian violet for general staining purposes, as defined by the Commission 
on the Standardization of Biological Stains, must be either penta-methyl or hexa- 
methyl pararosanilin, or else a mixture of methylated pararosanilins composed 
primarily of the two compounds just named and having a shade at least as deep 
as that recognized in the trade as methyl violet 2B. In other words the dye must 
be Colour Index Xo. 680 or No. 681. 

4. Gentian violet samples to be certified by the Commission must be of at least 
75 per cent dye content as determined by reduction of TiCl3 in an atmosphere of 
carbon dioxide. The diluent used must be dextrin. 

5. The sample of crystal or gentian violet should prove satisfactory for staining 
bacteria according to the Gram technic when made up in a 0.5 per cent aqueous 
solution, without the use of anilin oil or any other mordant, and stained by the 
following procedure: crystal or gentian violet 1 minute, Lugol's iodine solution 1 
minute, 95 per cent alcohol 30 seconds, 0.05 per cent safranin solution 10 seconds. 
In making the test, a weakly Gram-positive orgnism and a Gram-negative organism 
should be tested. 

6. The sample should prove satisfactory in the Flemming triple stain when 
used with safranin and orange G. For this purpose it must be tested by histologists 
skilled in the technic in question. It should also prove a satisfactory nuclear 
stain in general histology. 

7. Either gentian violet or crystal violet for certification must have sufficient 
bacterostatic power so that, when added to nutrient agar in the proportion of one 
part to a million, it will entirely prevent the growth of Bacillus suhtilis when this 
organism is streaked over the surface of the hardened agar. It must be understood, 
however, that this bacterostatic property of the dye is still under investigation 
and has a relation not yet fully understood to the control of certain diseases. As 
new investigations bring new light on the subject it may be necessary to draw up 
new specifications to cover this point. 

SPECIFICATIONS FOR PYRONIN 

1. Pyronin designed for staining purposes must be either pyronin G (Colour 
Index No. 739) or pyronin B (Colour Index No. 741). The container in which it is 
sold should be marked plainly as to which of these two dyes is furnished. 

2. Samples of pyronin G must be characterized by an absorption curve showing 
a maximum absorption at about oiSfifx Avhen determined in a 0.002 per cent solution 
in a layer 1 milimeter thick. To be certified by the Commission these samples 
should be of such a strength that the extinction coefficient at the point of maximum 
absorption shall be not less than 1.20. 

3. Samples of pyronin B tested in the spectrophotometer under the above 
mentioned conditions must have an absorption maximum at about 550;a/x, and must 
be of such a strength that the extinction coefficient at the point of maximum ab- 
sorption shall be not less than 1.00. 

4. The samples shall be satisfactory for use in the Pappenheim combination 
with methyl green. In the case poor results are obtained with the formulae for 

135 



this stain commonly found in the literature it should be suspected that the trouble 
might be due to the greater concentration of pyronins now available and the tests 
should be made by the following formula calling for a suialler proportion of pyronin : 

Methyl green 100 gm. 

Pyronin 25 gm. 

Alcohol o. cc. 

Glycerin 20 cc. 

,2 per cent carbolized water 100 cc. 

SPECIFICATIONS FOR EOSIN Y 

1 . vSamples of eosin Y must be of the type represented by Colour Index No. 768. 
They must, furthermore, be the dibasic sodium salt of tetra-brom fluorescein 
having an absorption maximum at approximately 516^;* as determined in a .002 
per cent solution in a layer 1 cc. thick in a spectrophotometer. They should be 
readily soluble in water and alcohol (insoluble material not over 0.5 per cent) . 

2. Eosin samples to be certified by the Commission must contain at least 85 
per cent total color as determined when reduced by titanous chloride in an atmos- 
phere of carbon dioxide. 

3. Eosin samples must yield a satisfactory Wright's stain in combination with 
methylene blue by the formula given in Mallory and Wright (1924) page 470. 
The quality of the resulting compound d\'e must be judged by someone skilled in 
its use for staining blood. 

4. The samples must prove satisfactory for counterstaining against basic dyes 
in histological technic, their performance to be judged by experts in the staining 
procedures involved. 

5. Eosin should prove satisfactory with methylene blue in the Levine eosin- 
methylene-blue medium for the differentiation of certain organisms of the colon 
typhoid group. In making this test the method should be followed wliich is 
published by Levine (1921) p. ()2-4. 

SPECIFICATIONS FOR ORANGE G 

1. Samples of orange G submitted for certification must be of the t^^pe listed in 
the Colour Index as No. 27 and must be characterized by an absorption maximum 
at approximately 485/A/i as determined in a .004 per cent solution in a layer 1 cc. 
thick in a spectrophotometer. 

2. The total dye content of the samples must be at least 80 per cent as de- 
termined by reduction of titanous chloride in an atmosphere of carbon dioxide. 

3. The samples must prove satisfactory for counterstaining in histology and 
cytology, giving a clear orange shade and in a brown tone to the cytoplasmic bodies. 
They shall also be satisfactory for the Flemming triple stain, in which this dye is 
used in contrast to safranin and crystal violet. Its performance in these procedures 
shall be judged only by ones skilled in the technic in question. 

SPECIFICATIONS FOR HAEMATOXYLIN 

1. A sample of haematoxylin to be considered for certification must consist of 
well defined crystals showing sandy or light brown color and shall contain less than 
0.1 per cent ash. With NaOH it shall yield a purple solution turning brown on 

1S6 



standing in presence of the air; with lead acetate it shall yield first a colorless and 
then a blue precipitate darkened by the air. 

2. When used by one of the standard methods such as that of Haidenhain for 
staining actively growing root tips or other good cytological material showing 
mitosis, it shall give a sharp, vigorous black staining of the chromosomes. When 
used in the Delafield method, it must give a clear-cut blue picture of chromatic 
material. These tests shall be made by someone skilled in the technic involved. 

3. Solutions shall show no discoloration and shall retain their staining qualities 
upon standing for a period of three or four weeks. 

SPECIFICATIONS FOR THIONIN 

1. Samples of thionin (Syn. Lauth's violet) must correspond to Colour Index 
No. 920 and be characterized by an absorption curve Avith a maximum at about 
aOififi as determined in a ,001 per cent solution in a layer 1 cc. thick in a spectro- 
photometer. 

2. Samples submitted for certification must have a dye content of at least 
85 per cent as determined by titanous chloride reduction in an atmosphere of 
carbon dioxide. 

3. The samples should prove satisfactory for staining frozen sections of fresh 
animal tissue and should show good metachromatic effects when applied in a 1 
per cent solution for 1 to o minutes, followed by rapid washing and mounting in 
water. Their performance in this matter shall be judged by someone familiar with 
the technic. 

4. The samples should prove satisfactory for staining bacteria by the "little 
plate" technic described by W. D. Frost in Jr. Inf. Dis. 19, (1916) p. 273-287 



137 



APPENDIX III 
BIBLIOGRAPHY 

(Matter in parenthesis indicates the purpose for which each reference is cited in the 
preceding pages, not necessarily the main subject matter of the article in question.) 

Anonymous. 

1865. Injectionsmassen von Thiersch und MuUer, Arch. Mikr. Anat., 1, llfS. 
(Use of carrainates with oxalic acid.) 
Albert, H. 

1920. Diphtheria bacillus stains with a description of a "new" one. Am. J. 
Pub. H., 10, 334- (Toluidin blue and methyl green for staining 
diphtheria preparations.) 
Ambler, J. A., and Holmes, W. C. 

1924. The investigation of biological stains in the Color Laboratory of the 
Bureau of Chemistry. Sci., 60, 501-502. 
Benda, Carl. 

1891 . Neue Mittheilungen uber die Entwickelung der Genitaldriisen and uber 
die Metamorphose der Samenzellen. Arch. f. Anat. u. Phys. {Phys. 
Aht.) 1891, 5Jf9-552. (Safranin with light green for staining 
spermatozoa.) 
1899. Weitere Mittheilungen liber die Mitochondria. Arch.f. Anat. w. Phys. 
{Phys. Aht.) 1899,376-383. (Proposes crystal-violet-alizarin method 
for chondriosomes.) 
1901. Die Mitochondriafarbung und andere Methoden zur untersuchung der 
Zellsubstanzen. Anat. Anz., Ergdnzhft. 19, 155-17Jf. (Describes the 
crystal- violet-alizarin method.) 
Beneke. 

1862, Correspbl. d. Ver.f. gemeinsch. Arheiten, No. 59, 980. (A note without 
title, being first reference to use of anilin dyes in histology.) 
Bensley, R. R. 

1911. Studies on the pancreas of the guinea pig. Am. J. Anat., 12, 297-388. 
(Acid fuchsin and Janus Green for chondriosomes. Describes 
"neutral gentian.") 
Bergonzixi, C. 

1891. tjber das Vorkommen von granulierten basophilen und acidophilen 
Zellen im Bindegewebe und uber die Art. sie sichtbar zu machen. 
Anat. Anz., 6, 595-600. (Methyl orange in place of Orange G in 
Ehrlich-Biondi stain.) 
Bernthsen, a. 

1885. Studien in der Methvlen blau gruppe. Liebig's Ann. de. Chimie., 230, 

73-136, 137-211. ^(Chemistry of Azur I, etc.) 
1906. Ueber die chemische Natur des Methylenazurs. Ber. d. Deut. Chem, 
GesselL, 39, II, 1804--1809. 
Best, F. 

1906- Ueber Karminfarbung des Glycogens und der Kerne. Zts. Wis. Mikr., 
23, 319-322. 

BOHMER, F. 

1865. Zur pathologischen anatomic der Meningitis cerebromedullaris epi- 
demica. Aerztl. Intelligenzb. {Munich). 12, 539-548. (First use of 
alum haematoxylin.) 

BOTTCHER, A. 

1869. Ueber Entwickelung und Ban des Gehorlabyrinths nach untersuchungen 
an Saugethieren. I Theil. Verh. Kais. Leop.-Carol. deut. Akad. 
Naturf., Dresden, 35, Abh. No. 5, pp. 1-203. (First use of alcohol for 
differentiation after staining.) 

138 



Brasil, Louis. 

1905. Sur la Reproduction des Gregarines monocystidees. Arch, de Zool. 

Exper. et Gen., ^ Ser., 4, 69-100. (Light green with haematoxylin for 
sections of seminal vesicles.) 
Browning, C. H., Gilmore, M., and Mackle, T. J. 

1913. The isolation of tjTjhoid bacilli from feces by means of brilliant green 
in fluid media. J. Hyg., 13, 33o-3It2. 

Carnoy, J. B., and Lebrux, H. 

1897. La fecondation chez I'Ascaris megalocephala. La Cellule, 13, 63-195. 

(Congo red with haematoxylin and other nuclear stains.) 

Chamberlain, C. J. 

192-i. Methods in Plant Histology. Fourth edition, xi and 3^9 pp. Univ. of 
Chicago Press. 

ClACCIO, C. 

1906. Rapporti istogenetici tra il simpatico e le cellule cromaffini. Arckivio. 

Ital. di. Anat. e. d. Embriol , 5, 2-56-267. (Iodine green with acid 
fuchsin and picric acid for nervous tissue.) 

Conn, H. J. 

1921. Rose bengal as a general bacterial stain. /. Bad., 6, 253-25^. 
CORTI, A. 

1851. Recherches sur I'organe de I'ouie des mammiferes. Zts. Wis. Zoo!., 3, 
109-169. (An earlv use of carmin in histologv, see note 10, p. 143-4). 
Cyon, E. 

1868. L'eber die Xerven des Peritoneum. Ber. d. k. Sachs Gessel. d. Wiss. zu 
Leipzig., 20, 121-127. (Gives technic of carmin staining.) 

CZOKOR, J. 

1880. Die Cochenille-Carmin losung. Arch. Mik. Anat., 18, ^12-Ifl4- (Uses 
alum cocchineal.) 
Daddi, L, 

1896. Xouvelle Methode pour colorer la graisse dans les tissues. Archives 
Ital. de Biol, 26, H3-146. (Proposes Sudan HI.) 
V. DiGRALSKi, W., and Coxradi, H. 

1902. Ueber ein Verfahren zum Nachweis der t>T)hus bazillen. Zts. f. Hyg. 
39, 283. (Uses crystal violet in agar.) 

Ebbinghaus, Heinr. 

1902. Eine neue Method zur Farbung von Hornsubstanzen. Ctblt.f. Allgem. 
Path. u. Path. Anat., 13, ^22-426. (Methyl orange for keratin.) 

Ehrlich, P. 

1910. Enzyklopadie der Mikroskopischen Technik. Second Edition. Urban & 
Sckwartzenberg, Berlin. Vol. 1, 800, pp.; 2, 680, pp. 

Ehrltch, p., and Lazarcs, A. 

1898. Die Anaemic. I Abt. In NothnageVs Spec. Path. u. Ther., Bd. 8, 

Vienna. (Describes various "neutral" stain mixtures; the "triacid" 
mixture; also pyronin and narcein with methyl green or methylene 
blue; and narcein with acid fuchsin and methyl green.) 

Ehrenberg, C. G. 

1838. Die Infusionsthierchen als volkommene Organismen. Leipsig. 
Endo, S. 

1904. Ueber ein Verhaften zum Nachweiss der Typhusbacillen. Cenibl. f. 
Bakt., I Abt., Orig., 35, 109-110. (Proposes the fuchsin agar known 
as "Endo medium.") 

Faris, H. a. 

1924. Neutral red and Janus green as histological stains. Anat. Rec, 27, 
2U-2U- 
Fischel, Alfred. 

1901. Untersuchungen liber Vitale Farbung. Anat. Hefte. I Abt. Bd. 16, 
No. 3ik {Hjte. 52/3) U7-519. (Auramin for salamander larvae.) 

139 



Flemming, W. 

1881. Ueber das E. Herraann'sche Kernfarbungs verfahren. Arch. Mikr. 

Anat., 19, 317-330. (Magdala red as a nuclear stain. Investigated 
principle of differentiation with alcohol.) 

1884. Mittheilungen zur Farbetechnik. Zts, Wis. Mikr., 1, 349-361. 

1891. Ueber Theilung und Kernformen bei Leukocyten, und iiber denen 
Attractionsspharen. Arch. Mik. Anat., 37, ^49-298. (Triple staining 
technic — gentian violet, safranin and orange G — described on p. 296.) 
Foot, Katherine and Strobell, Ella Church. 

1905. Prophases and Metaphases of the first maturation spindle of Allolobo- 
phora foetida. Am. J. Anat., 4, 199-2If3. (Use of Bismarck brown 
for staining chromosomes in smear preparations of eggs.) 
Frey. 

1868. Die Hamatoxylin farbung. Arch. Mikrosk. Anat., 4, 3^5-6. (First 
use of alum and haematoxylin in a single solution.) 
Frost, W. D. 

1916. Comparison of a rapid method of counting bacteria in milk with the 

standard plate method. ./. Inf. Dis., 19, 273-287. (Use of thionin 

for staining young bacterial colonies.) 
Gerlach, J. 

1858. Mikroskopische Studien aus dem Gebiet der menschlichen Mor- 
phologic. Erlangen, 1858. pp. 72. (Shows the advantage of dilute 

carmine solutions.) 
Giemsa, G. 

1902. Farbemethoden fiir Malariaparasiten. Centbl. f. Bakt., I Abt., 32, 

307-313. (Describes use of Azur I and Azur II.) 
1902. Farbemethoden fiir Malaria parasiten. Centbl. f. Bakt., I Abt., 31, 

.'f29-J^30. (Showing the value of preparing blood stains with eosin 

and Azur I alone.) 

1904. Eine Vereinfachung und Vervollkommnung meiner Methylen-azur- 

Methelen-blau-eosin Farbemethode zur Erzielung der Romanowsky- 
Nochtschen Chromatinfarbung. Centbl. f. Bakt., I Abt., 37, 308-311. 
Gierke. H. 

1884, 1885. Farberei zu mikioskopischen Zwecken. Zts. Wis. Mikr., 1, 
62-100, 372-m, m-557, 2, 13-36, 16^-221. (Discussion of history 
of staining.) 
Goppert, H. R., and Cohn, F. 

1849. Ueber die Rotation des Zellinhaltes von Nitella flexilis. Botan. Zeitg., 7, 
665-673, 681-691 , 69 7-705, 713-719. (First use of carmin for micro- 
scopic staining purposes.) 
Gr.\m, C. 

1884. Ueber die isolierte Farbung der Schizomyceten in Schnitt imd 
Trochenpraparaten. Fortschritte der Med., 2, 185-189. 
Grenacher, H. 

1879. Einige Notizen zur Tinctionstechnik, besondes zur Kernfarbung. 

Arch. Mikr. Anat., 16, Ji63-J^71. (Uses alum carmin.) 
Griesbach, H. 

1882. Ein neues Tinctionsmittel fiir menschliche und thierische Gewebe. 

Zool. Anz. 5, 406-^10. (Iodine green as a nuclear stain.) 

1880. Weitere Untersuchungen iiber Azofarbstoffe behufs Tinction mensch- 

licher und thierischer Gewebe. Zts. Wis. Mikr., 3, 358-385. 
(Congo red and amaranth for staining axis cylinders.) 

GUYER, M. F. 

1917. Animal Micrology. Revised Edition. 289 pp. University of Chicago 

aTCSS 
IL\NSEN, F. C. C. 

1905. Ueber Eisenhamatein, Chromalaumhamatein, Tonerdealaunhamatein, 

Hamateinlosungen und einige Cochenille farblosungen. Zts. Wis. 
Mikr., 22, 45-90. 

140 



Hartig, Th. 

1854. Ueber die Functionen des Zellenkerns. Botan. Zeitg , 12, o7If-58It. 

(A comprehensive investigation of the ability of various parts of 

plant protoplasm to take carmin.) 
1854. Ueber das Verhalten des Zellkerns bei der Zellentheilung. Botan. Zeitg., 

12, 893-902. (An early use of carmin.) 
1858. Entwickelungsgeschichte des Pflanzenkeims, dessen stoffbildung 

wahrend der Vorgange des Reifens und des Keiraens. Leipsig, 1858, 

16Jt pp. (Uses carmin.) 

Hbidenhain, R. 

1888. Beitrage zur Histologic und Physiologic der Diinndarmschleimhaut. 
Pflug. Arch. Ge.s. Physiol, 43, Supple., 103 pp. 

Hebmann, E. 

1875. Ueber eine neue Tinctionsmethode. Tagbl. d. IfS Versaml. deut. Naturf. 
u. Aerzte, Graz 1875, 105 pp. (Early use of alcohol differentiation 
to bring out nuclei.) 

Hjcksox, S. J. 

1901. Staining with Brazilin. Qu. .J. Micr. Sci., N. S. 44, J^69-J^71. iBrazUin 
after iron alum.) 
HucKER, G. J. and Conts, H. J. 

1923. Methods of Gram staining. .V. Y. Agric. Exper. Sta. Tech. Bid. 93. 
Jenner, Louis. 

1899. A new preparation for rapidly fixing and staining blood. Lancet, 1899, 

Pf. 1. 370. 
Kehrmaxx, F. 

1906. Ueber Methylen-azur. Ber. d. Deut. Chem. Gessell., 39, II, 1W3-1W8. 
Klbbs, G. 

1886. I.Teber die Organisation der Gallerte bei einige Algen und Flagellaten. 
Unter. Bot. Inst. Tubingen. 2, No. 2, 333-^18. (Congo red as reagent 
for cellulose: see p. 369.) 

KULTSCHITZKY, N. 

1895. Zur Frage uber den Ban der Milz. Arch. Mik. Anat., 46, 673-695. 
(Magdala red for elastic tissues.) 
Lee, A. B. 

1921. The Microtomists Vade-mecum. Eighth Edition, edited by .J. B. 
Gatenhy. Blahistons, Philadelphia. 
Lee, a. B., and Mayer, P. 

1907. Grundzuge der mikroskopischen Technik. Third Edition . Berlin. 1907. 
Leishmanx, W. B. 

1901. A simple and rapid method of producing Romanowsky staining in 
malarial and other blood films. Brit. Med. J., 1901, Pt. 2, 757-758. 
(Redissolved precipiate of Xocht stain in methyl alcohol.) 
Levine, M. 

1921. Bacteria fermenting lactose and their significance in water analysis. 

Iowa Engineering Exp. Sta. Bui. 62. (Use of eosin-methylen-blue 
agar; see p. 62-4.) 
List, J. H. 

1885. Zur Farbetechnic. Zt^s. Wis. Mikr., 2, lJi^5-156. (Uses eosin preceding 
methyl green.) 
MacNeal, W. J. 

1906. Methylene violet and Methylene azur. ./. Inf. Dis., 3, U2-It33. 
(The history of blood stains and the chemistry of its ingredients.) 

1922. Tetrachrome blood stain; an economical and satisfactory imitation of 

Leishmann's stain. J. Am. Med. Assn., 78, 1122. 
1925. Methylene violet and methylene azure A and B. ./. Inf. Dis., 36, 
538-5^6. 
Maijx)ry, F. B. 

1900. A contribution to staining Methods. J. Exp. Med., 5, 15-20. (Anilin 

blue connective tissue stain described.) 

141 ^ 



1904. Scarlet fever. Protozoan-like bodies found in four cases. /. Med. Res., 
10, Jf83-Jt92. (Eosin as contrast stain to methylene blue for tissue, 
especially in pathology.) 
Mallory, F. B., and Wright, J. H. 

1924. Pathological Technic. Eighth Edition. Saunders, Philadelphia. 
Mann, Gustav 

1902. Physiological Histology. Clarendon Press, Oxford. 
Maschke, O. 

1859. Pigmentlosung als Reagenz bei Mikroskopisch phj^siologisch XJnter- 
suchungen. Bot. Zeitg., 17, 21-27. J. f. prakt Chem. v. Erdmannu. 
Wether, 76, 37. (First use of indigo.) 
Mayer, Paul. 

1878. Die Verwendbarkeit der Cochenille in der microscopischen Technic. 
Zool. Anz., 1, 3If5~6. (Uses Cocchineal with alura.) 

1891. Ueber das Farben mit Hamatoxylin. Mitt a. d. Zool. Stat. Neapel, 10, 

170-186. (Haemalura, haemacalcum, etc.) 

1892. Ueber das Farben mit Carmin, Cochenille und Hamatein Thonerde. 

Mitt. a. d. Zool. Stat. z. Neapel, 10, J^80-504. 
1896. Ueber Schleimfarbung. Mitt. a. d. Zool. Stat. z. Neapel, 12, 303-330. 
(Describes muci-carmine, muc-haematin and gluchaematin.) 

1899. Ueber Hamatoxvlin, Carmin, and verwandte Materien. Ztsch. Wis. 

Mik., 16, 196-220. 

McClung, C. E. 

1923. Haematoxylin. Sci., 58, 51'. 

MiCHAELIS, L. 

1900. Die vitale Farbung, eine Darstellungsmethode der Zellgranula. Arch. 

Mikr. Anat., 55, 558. (Uses Janus green for chondriosomes.) 

1901. Ueber Fett Farlistoffe. Virchows Arch. J. Path. Anat. u. Phys., 164. 

263. (Proposes Sudan IV) . 
MiJLLER, H. A. C. 

1912. Kernstudien an Pflanzen. Arch. f. Zelljorschiing. 8, Hft. 1, 1-61. 
(Applies to plant pathology the stain mixture of Pianese — malachite 
green, acid fuchsin and martins yellow.) 

NOCHT. 

1898. Zur Farbung der Malariaparasiten. Centbl. f. Bakf. I Abf., 24, 839-843. 

(First to polychromize methylene blue intentionally in preparing 
blood stains.) 

Osborne, S. G. . 

1857. Vegetable cell structure and its formation as seen in the early stages of 
the growth of the wheat plant. Trails: Micro. Soc, 5, 10^-122. 
(Observes coloring of cell contents in plants grown in colored solu- 
tions — carmin. indigo, or vermillion.) 

Pal.\dino, Giovanni. 

1895. Delia nessima partecipazione dell' epitelio della mucosa uterina e della 
relative glandole alia formazione della decidua vera e riflessa nella 
donna. Rend. d. Accad. Sci. fisiche e matemat. Napoli, 34, p. 208-S15. 
(Biebrich scarlet with alum haematoxylin.) 

Pappenheim, a. 

1899. Vergleichende Untersuchungeniiber die elementare Z usammensetzung 

des rothen Knochenmarkes einige Saugethiere. Virchow\s Arch. /. 
Path. Anat. v. Phys.. 157, 19-76. (Uses mixture of methyl green 
and pyronin.) 

Pelagetti, M. 

1904. Ueber einige neue Farbungsmethoden mit Anwendung der Zenkerschen 
Fi.xierungsfliissigkeit in der histologischen Technik der Haut- 
Monatsch. f. Prak. Dcrmat., 38, p. 532-536. (Biebrich scarlet after 
polcyhrome methylene blue or after Unna's haematein. Rose bengal 
following haematoxylin.) 

142 



Peter, Karl. 

1899. Die Bedeutung der Nahrzelle im Hoden. Arch. Mirk. Anai., 53, 180- 
211. (Light green with haematoxj'lin.) 

PfITzer, E. 

1883. Ueber ein Hartung und Farbung vereiniges Verfahren fur die unter- 
suchimg des plasmatischen zelleibs. Ber. Deui. Boi. Gesel., 1, hk-\7. 
(Picro-nigrosin for chromatin.) 

PlANESE, G. 

1896. Beitrag zur Histologic und Aetiologie des Carcinoms. Beitrag zur 
Path. Anat. u. AUgem. Path., Suppl. I, 193 pp. (Malachite green 
and martins yellow with acid fuchsin.) 

Phenani, a. 

1902. Contribution a I'etude de la ciliation. Arch. (T Anat. Micro., 5. (Light 
green with haematoxylin.) 

Raniver. 

1868. Technique microscopique. Arch. de. Phi/s. 1, Xo. 2, 319-321; No. 5, 
666-670. (First use of picro-carmin in a single procedure.) 

Rawitz, B. 

1899. Bemerkungen uber Karminsaure und Hamatein. Anat. Anz., 15, 
437-Ui. 
REtiTER, Karl. 

1901. Uber den farbenden Best andteil der Romanowsky-Xochtschen Malaria 
plasmodien farbung, seine Reindarstellung und praktische Verwen- 
dung. Centhl.f. Bakt. I Abt., 30, ^45-^-56'. (Dis.solves precipitate of 
Nocht stain in absolute alcohol plus anilin oil.) 
Robertson, O. H. 

1917. The effects of experimental plethora on blood production. J. Exp. 
Med., 26, 221-237. (Use of brilliant cresyl blue for staining reticu- 
lated blood cells.) 
Robinson, H. C. and Rettger, L. F. 

1916. Studies on the use of brilliant green and a modified Endo's medium in 
the isolation of Bacillus tNphosus from feces. ./. Med. Re.^. N. S.. 29, 
363-376. 

ROMANOV.SKI. D. L. 

1891. On the question of parasitology and therapy of malaria (In Russian). 

Imp. Med. Mililari/ Acad., ^ Dissert. No^ 38, St. Petersburg, 1891. 

(Proposes combination of eosin and mthylene blue for staining blood.) 
1891. Zur Frage der Parisitologie und Therapie der Malaria. St. Petersb. Med. 

Wochenschr.. 16, 297-302, 307-315. A slightly condensed version of 

the above. 

Rothberger, C. J. 

1898. Differential diagno.«tische unersuchungen mit gefarbten Xahrboden. 
Centbl. f. Bakt. I Abt., 24, 513-518. (Neutral red as indicator in 
media for differentiating typhoid and colon bacilli.) 

SCHAFFER. 

1888. Die Farberei zuni Studium der Knochenentwicklung. Zcit. Wis. Mikr., 
5, 1-10. (Fast yellow for bone; Congo red for embryo sections.) 

SCHULTZ. G. 

1923. Farbstofftabellen. 6 Aufl. Berlin. Bd. 1, 385 pp; Bd. 2, 290 pp. 

Schwartz, E. 

1867. Ueber cine Methode doppelter Farbung Mikroskopischer Objecte und 
ihre Anwendung, etc. Sitz. berichte d. k. Acad. d. Wiss. Wien Bd. 55, 
Eft. 1, 671-689. (First double staining.) 

Schweiger-Seidel, F., and Dogiel, J. 

1866. Ueber die peritoneal Hiille bei Froschen und ihren zusammenhang mit 
dem Lymphagefassysteme. Ber. d. k. Sachs. Gessel. d. Wiss. zu 
Leipzig, 18, 2Jt7-25If. (Use of carminates Avith acetic acid.) 

143 



Scott, R. E., and French, R. W. 

1924a. Standardization of Biological Stains. Mil. Surg., Aug. 192Jf., 15 pj). 
1924b. Standardization of Biological Stains. II Methylen blue. Mil. Sura., 

Sept. 192J,, 16 pp. 
1924c. Standardization of Biological Stains. III. Eosin and haematoxylin. 
Mil Surg., Nov. 1924 > 8 pp. 
Smith, Louise. 

1920. The hypobranchial apparatus of Spelerpes bislineatus. J. Morph., 33, 

527-583. (Use of methylene blue in staining cartilege by the Van 
Wijhe technic.) 
Society of Dyers and Colourists. 

1924. Colour Index. Edited by F. M. Rowe. Publi'ihed by the Society, 
Bradford, Yorkshire, England. 
Spulek, a. 

1901. Ueber eine neue stiickfarbemethode. Deut. Med. Wchnschr., 2*7 y 
Vereine-Beilage No. 14, 116. (Iron alum cocchineal.) 
Stilling, H. 

1886. Fragmente zur Pathologie der Milz. Virchows Arch. f. Path. Anal. u. 
Physiol. 103, 15-88. (Iodine green for amyloid). 
Teichmuller, W. 

1899. Die eosinophile Bronchitis. Deut. Arch. Klin. Med., 63, -^^^-ffSS. 
(Eosin for staining sputum, followed by methylene blue.) 

TORREY, J. C. 

1913. Brilliant green broth as a specific enrichment medium for the para- 

t%'poid-interiditis group of bacteria. J. Inf. Dis., 13, 268-72. 
Unna, p. G. ' 

1891. Ueber die Reifung unserer Farbstoffe. Zts. Wis. Mikr., 8, ^75-^7. 
(Polychrome meth^dene blue). 

1921. Chromolyse. Abaerhaldens Handb. der Biol. Arbeitsmethoden. Aht. 6, 

Tcil 2, Hft. 1, {Liefng. 17) 1-62. 
Vaughan, R. E. 

1914. A method for the differential staining of fungus and host cells. .-Inn. 

Mis. Bof. Gard. 1, 21^.1-21^2 . (An adaption for botanical purposes of 
Pianese's staining malachine green acid fuchsin and martins yellow.) 
Vinassa, E. 

1891. Beitrage zur pharmakognostischen Miki'oskopie. Zts. Wis. Mikr., 8 
(Auramin for plant sections.) 
Waldeyer. 

1 864. Untersuchungen iiber den Ursprung und den Verlauf des Axsencylinders 
bei Wirbellosen und Wirbelthieren sowie iiber dessen Endverhalten 
in der quergestreiften Muskelfaser. TIenle & Pfeifers Zeitschr. f. 
rationelle. Med. 3 Reihe. Bd. 20, 193-256. (First attempt to stain with 
logwood extract. Early use of anilin dj'cs). 
Weigert, Carl. 

1881. Zur Technik der mikroskopischeu Bakterien untersuchungen. Vir~ 
chow's Arch. f. Path. Anat. u. Phys., 84, p. 275-315. (Gentian violet 
for fibrin and Neuroglia.) 
1898. Ueber eine Methode zur Farbung elastischer Fasern. Centbl. f. Allgeni. 
Path. Anat., 9, 289-292. (Fuchsin for elastic tissue). 
Williams, B. G. R. 

1923. Cresylecht violet, a rare dve. Jr. Lab. & Clin. Med., 8, No. J, Jan. 

1923. i pp. 
Wixogradsky. 

1924. Sur Fetude microscopique du sol. C. R. Acad. d. Sci. 179, 367. (Ery- 

throsin for staining bacteria in soil.) 

ZiMMERMANN, A. 

1893. Beitrage zur Morphologic und Physiologic der Pflanzenzelle Bd. IT. 
Tubingen, 1893. 35 pp. (Iodine green as chromatin stain for plant 
cells.) 

ZSCOKKE, E. 

1888. Ueber einige neue Farbstoffe beziiglich ihrer Verwendung zu histofo- 
gischen Zwecken. Zts. Wis. Mikr., 5, .^65-^70. (Benzopurpia in 
contrast to haematoxylin.) 

144 



REPORTS OF COMMISSION ON 
STANDARDIZATION OF BIOLOGICAL STAINS 

AND OF 

RELATED COMMITTEES 

Com. on Bact. Technic, of Soc. Amer. Bacteriologists, H. J. Couii, chairman. 
1921. The Production of Biological Stains in America. Science, 53, 289-290. 
1922a. An Investigation of American Stains. J. Bad., 7, 127-li8. 
1922b. An Investigation of American Gentian Violets. J. Ba£t., 7, 529-536. 
Com. on Standardization of Bioi.. Stains, of Nat. Res. Council, H. J. Conn, 
chairman. 
1922a. The Standardization of Biological Stains, Science, 55, |J-4i- 

American Biological Stains compared with those of Griibler. Science, 

55, 28^-285. 
Preliminary Report on American Biological Stains. Science, 56, 

156-160l 
Collaborators in the Standardization of Biological Stains. Science, 
56y 59Jt-596. 
Commission on Standardization of Biol. Stains. H. J. Conn, Chairman. 
1922.a The Present Supply- of Biological Stains. Science, 56, 562-563. 
American Eosins. Science, 56, 689-690. 
The preparation of Staining Solutions. Science. 57, 15-16. 
Safranin and Methyl Green. Science, 57, 30^-305. 
Thionin. J. Dairy Science, 6, 135-136. 

Dye Solubility in Relation to Staining Solutions. Science, 57, 638-639. 
Standardized Nomenclatures of Biological Stains. Science, 57, 638-639. 
Certified Methylen Blue. Science, 58, 'il-/t2. 
Investigations Concerning Imported Biological Stains. Science, 54, 

328-331. 
Certified Safranin. Science, 54, 556-557. 
A report on Basic Fuchsin. Science, 60, 378-388. 
Certified Stains— What Thev Are and How to Obtain Them. ./. Lab. 

and Clin. Med., 10, 321-322. 
New Applications of Biological Stains. J. Chein. Education, 2, 18'^- 
185. 



1922b. 



1922.C 
1922d. 



1922b. 

1923b. 

1923c. 

1923d. 

1923e. 

1923f. 

1923g. 

1924a. 

1924b. 
1924c. 
1925a. 

192ob. 



145 



INDEX 



In this index the d^'es named are printed either in bold-faced type or italics; 
preferred designations are in bold-faced type, synonyms in italics. Figures in 
bold-faced type indicate the principal references. 



Absorption curv^es, 28-31 

Acid bordeaux, 35 

Acid dyes, 16 

Acid fuchsin, 22, 32, 64, 118, 134 

Acid green, 62 

Acid magenta, 64 

Acid orange, 37 

Acid rubin. 64 

Acid yelloiv, 36, 41 

Acid yellow D, 36 

Alcohol soluble eosin, 79, 8U 

Algae, .staining of, 57, 73, 81 

Albert stain, 50, 115 

Alizarin, 22, 42, 91 

Alizarin carniin, 43 

Alizarin No. 6, 43 

Alizarin orange, 41 

Alizarin jpurpurin, 43 

Alizarin red, water sol., 43 

Alizarin red S, 43, 1 1 3 

Alizarin sulphate, 43 

Alizarin yellow R, 41 

Alizarin yellow GG, 41 

Altmann, 65 

Amaranth, 37, 112 

Ambler, 52 

Amethyst violet, 56 

Amido-azins, 22, 53-55 

Ammonium bases, 15 

Ammonium picrate, 32 

Amyloid, staining of, 66, 67, 71 

Andrade indicator, 65, 119, 134 

Anilin, 11, 14 

Anilin blue, ale. sol., 71 

Anilin blue, W. S., 22, 72, 121 

Anilin dyes, first use of, 9 

nature of, 1 1 
Anilin red, 63 
Anthracene yellow, 41 
Anthracene yelloiD RN. 41 
Apathy, 97, 127 
Archelline 2B, So 
Ascaris eggs, staining of, 61 
Auramin, 22, 60, 117 
Aurantia, 33, 110 
Aurin, 74 
Aurin R, 74 
Auxochromes, 14, 15 
Axis cylinders, staining of, 37, 40, 73 
Azidine blue SB, 41 
Azin radical, 17, 53 



Azins, 22, 53-57 
Azobenzene, 17 
Azo-bordeaux, 35 
Azo dyes, 17, ^1, 33-41 

radical, 17 
Azo rubin, 37 
Azure I, 47, 48 
Azure II, 49, 89 
Azure A, 48, 90 
Azure B, 48 

Bacillus subtilis, 135 

Bacteria, staining of, 46, 62. 68, 76, 

81, 83 
Bacteriostatic action of gentian violet, 

68, 69, 135 
Balch, 89 
Basic dves, 15 

Basic fuchsin, 22, 28, 63, 118, 1.33 
Basic rubin, 63 
Benda, 43, 5Q, 62, 68. 97, 98, 113. 116. 

117, 119, 128 
V. Beneden, 61, 1 17 
JocncKG 
Ben.slev, 35, 65, 70, 98, 111, 119, 128, 

134 
Bensley-C'owdrv technic, 65, 70, 119, 

120 
Bensley's neutral gentian, 34 
Benzamine blue 3B, 41 
Benzene, 11 
Benzene yellow, 41 
Benzene yellow PN, 41 
Benzo blue SB, 41 
Benzoin sky blue, 41 
Benzopurpin 4B, 40, 113 
Bergonzini, 36, 111 
Berlin blue, 72 
Bernthsen, 48, 49, 50, 56, 88 
Best, 93 

Biebrich scarlet, water sol., 39, 112 
Bindschedler's green, 44 
Bismarck brown G, R and GOOD, 39 
Bismarck brown Y 21, 39, 112 
Blood stains, 47, 49, 50, 54, Qo, 70, 71, 

88-90 
Bohmer, 9, 97, 127 
Bone, staining sections of, 36 
Bordeaux, 37 

Bordeaux B, BL, G, and R, 35 
Bordeaux SF, 37 



146 



Bordeaux red, 35, 111 
Hbttcher, 1) 
Brazalum, 96 
Brazil, 117 
wood, 9.) 
Brazilein, 96 
Brazilin, 95, 126 
Brilliant blue C, 51 
Brilliant cresyl blue, '-22, 51, 115 
Brilliant green, 22, 61, 117 
Brilliant pink\ 77 
Brom cresol green, 85 
Brom cresol purple, 85 
Brom chlor phenol blue, 85 
Brom phenol blue, 85 
Brora phenol red, 85 
Brom thymol blue, 85 
Broun salt R, -il 
Biitschli, 128 

Caesar red, 80 

Calcium salts, detection of, 43 

dauary yellow, GO 

Cancer tissue, staining of, 32, 61, do 

Capri blue, 53 

Carbinol, 19, 59 

Cardinal red, 41 

Carmin, 7, 8, 92, 124 

Carminic acid, 93, 125 

Carmin naphtha, 41 

Cartilage of frogs, staining of, 51 

Cellulose, staining of, 40, 62. 65 

Cerasin, 35, 41 

Cerasin red, 38 

Cerotin orayige, 41 

Chamberlain, 57, 81, 82, 116 

Champy-Kull technic, 33, 110 

China blue, 72 

Chlor azol blue 3B, 41 

Chlor phenol red, 85 

Chromatin, staining of, 50, 56, 67, 70, 

71, 93, 97 
Chrom black, 41 
Chromogens, 14, 15 
Chromolysis, 36, 54, 57, 70, 73, 94, 103 
Chromophores, 14, 15, 16-18 
Chrom violet, 74 
Chrysoin, 41 
Chrysoidin Y, 41 
Chrysoidin R, 41 
Ciaccio, 71, 120 
Cocchlneal, 92, 123 
Cohn, 8, 93 
Color, relation to chemical formula, 20 

relation to light absorption, 25 
Colour Index, 23 
Congo, 40 
Congo blue 3B, 41 
Congo red, 21, 40, 112 
Congo shy blue, 41 



Conn, 68, 123 

Connective tissue, staining of, 32, 34, 

65 
Copper, detection of, 97 
Corallin red, 22, 74 
Corallin yellow, 74 
Cortex, staining of, 65 
Corti, 8, 93 
Cotton blue, 72 
Cotton orange, 41 
Cotton red, 40 
Cotton red 4B, 40 
Cowdrv, 35, 65, 119, 120 
Cresyl blue 2RN and BBS, 51 
Cresyl echt violet, 52 
Cresyl red, 85 
Cresyl violet, 52, 115 
Croceinc scarlet, 39 
Crystal ponceau 6R, 41 
Crsytal violet, 21, 28, 67, 119, 134 
Cutinized tissues, staining of, 56 
Czokor, 92, 123 

Daddi, 38, 112 

Dahlia, Qo 

Dahlia B, 66 

Dark brown salt /?, 41 

Delafield, 80, 97, 127, 137 

Diamin red JfB, 40 

Diamond black F, 41 

Diamond f la vine, 41 

Diamond fuchsin, 63 

Diamond green, 61 

Dianil blue H3G, 41 

Dianil blue HOG, 41 

Dianil red ^C, 40 

Dianthin B, and G, 81 

Diazin green, 35 

Differentiation, first use of, 9 

Di-phenyl methane dyes, 60 

Diphtheria organism, staining of, 47, 

132 
Direct red, 40 
Direct red 4B, 40 
Dogiel, 9 
Double green, 69 
Double scarlet, 39 
Double staining, first use of, 9 
Dubreuil, 72, 121 
Duesberg, 68 
Dye, definition of, 15 

indexes, 23 
Dyes, classification of, 20 

nomenclature of, 22 

solubilities of, 24 

spectrophotometric analysis of, 25- 
31 

Ebbinghaus, 36, 111 
Ehrenberg, 7, 93 



147 



Ehrlich, 34, 36, 37, 52, 56, 57, 65, 66, 

70, 76, 77, 87. 88, 90, 07, 110, 120, 

121, 127 
Ehrlich-Biondi-Heidenhain stain. 34, 

36, 65, 70, 111, 118, 120 
Elastic tissue, 57, 62, 94 
Einbrvos, staining sections of. 35. 40, 

54, 72, 95 
Emerald green, 61 
Endo medium, 62, 63, 118, 134 
Eosin, alcoJiol soluble, 79, 80 
Eosin bluish, 80 

Eosiii BN, B, BW, and 7) AT, 80 
Eosin J, 81 
Eosin S, 80 

Eosin scarlet B and BB, 80 
Eosin W or WS {i. e. wafer soluble). 79 
Eosin Y, 79. 122, 136 
Eosinophile granules, 79 
Epithelium, staining sections of. 73 
Erythrocytes, 61 
Erythrosin, 22, 122, 81 
Erythrosin, B. R, and G, 81 
Erythrosin BB, 82 
Excelsior broicn, 39 
Ethyl eosin, 80 
Ethyl green, 61 
Extinction coefficient, 27 ' 
Eurhodins. 53-55 

Earis, 35, 54, 111, 115 

Vast acid green N, 62 

East blue 3R, 53 

East red, 37 

East red A or Ai', 41 

Fast red B or P, 35 

East red 0, 41 

Fast yellow, 35, 111 

Eat stains, 34, 38, 52 

Fettponceaii, 38 

Fibrin, staining of, 68 

Eischel, 60, 117 

Elemming, 9, 57, 68 

Elemming triple stain, 34, 56, 67, 102, 

110, 116, 119, 135, 136 
Eluorane, 78 

derivatiyes, 22, 78-83 
Fluorescein, 78 
Foot, 112 
French, 47, 48 
Erey, 9 

Frogs, staining cartilage of, 51 
Frost, 46, 114, 137 
Frozen sections, 67, 137 
Fuchsin, acid, 16, 22, 32, 64, 118, 134 
Fuchsin, basic, 9, 16, 19, 22, 28. 63, 

118, 133 
Fuchsin NB, 64 

Fuchsin S, SN, SS, ST, or .S77/, 64 
Fuchsinophile granules, 62 



Fungi, staining in sections of infected 
plants, 33 

Galeotti, 70, 120 

Galli, 73 

Gentian blue, 71 

Gentian violet, 22. 66, 67, 68, 119, 134 

Gerlach, 8, 9 

Giemsa, 49, 89, 114 

Gierke, 7 

Gold orange, 36, 37 

Gold yellow, 41 

Gonococcus, staining of, 54, 76, 70 

Goppert, 8, 93 

Graberg, 35, 111, 114 

Gram stain for bacteria, 39, 56, 68, 76, 

100, 101, 119, 135 
Gray R, B, BB, 57 
Grenadier, 93, 94. 124 
Griesbach, 37. 40. 77, 112, 113, 120, 

121 
(iriibler, 5, 89 

Ilaemahim, 97 

Haematein, 96, 126 

Haematoxylin, 9, 96, 126, 136 

Hansen, 92, 124, 127, 128 

Hartig, 8, 93 

Heidenhain, 35, 97, 127, 128, 137 

Held, 81, 122 

Ilelianthin, 3(5 

Heliotrope B, 5G 

Helvetia blue, 72 

Henneguy, 127 

Hermann, 9 

Hexamethyl violet, 67 

Hickson, 96, 126 

Hoffman green, 71 

Hoffman violet, 21, 65, 119 

Holmes, 52 

Hoyer, 124 

Huber, 73, 121 

Hucker, 68, 112, 116, 119 

Imperial yellow, 33 

Indamin radical, 17 

Indamins, 22, 44 

Indicators, 36, 40, 42, 43, 54, 65, 73, 

83-5, 95 
Indigo, 8, 91 
Indigo blue, 91 
Indigo-carmin, 92, 123 
Indigotinc la, 92 
Indin blue 2RD, 53 
Indulin black, 57 
Indulins, 22, 57 
Insects, staining tissue of, 46 
lodo-eosin B and G, 81 
Iodine green, 71, 120 
Iodine violet, 65 



148 



Iris violet, ofi 
Iron, detection nl". '.) 
Jsoruhin, 64 
/srael, 9.5, V2Q 

Janus green B, 35, 1 II 
Janus red 41 
Jarotsky, 57, 116 
Jenner, 89 
Juergens, G(i, 119 

Kaiser, 128 
Kehrraann, 48 
Keratin, staining of, 36 
Klebs, 40, 113 
Krumwiede, 61, 117 
Kiiltschitzky. 57, 116 

Lactone ring, 78 

Langerhans, islands of, 34 

Lauttis violet, 45 

Lazarus, 56 

Leather brown, 39 

Lefas, 71, 120 

Leishman, 89 

Leuco-base, 19, 59 

Leuco compounds, 18-20 

Leuco-fuchsin, 19, 62 

Levine, 47, 114, 136 

Light green, SF vellowish, 22, 62, 69, 

117 
Light green 2G, 3G, J^G, or 2GN, 62 
Light green N, 61 

Lignified tissue, staining of, 50, 56, 68 
List, 79, 122 

"Little plate" teclmic, 46, 137 
Litmus, 95 
Loeffler, 48 
Logwood, 9, 95 

Lorrain Smith fat stain, 52, 115 
Lyons blue. 71 

Maas, 61, 117 

MacNeal, 48, 49, 89, 90 

Magdala red, 22, 56, 81, 82, 116 

Magenta, 63 

Malachite green, 22, 32, 61, 117 

Malachite green G, 61 

Mallory, 79, 82, 89, 98, 105, 110, 114, 

119, 122 
Mallory connective tissue stain, 34, 65, 

73, 110, 118, 121, 134 
Manchester broum, 39 
Manchester yelloic, 32 
Mandarin G, 37 
Mann, 7, 72, 79, 121, 122 
Marine blue, 72 
Martius yellow, 32, 110 
Maschke, 8 
Mast cells, staining of, 66, 70, 76 



Mauveine, 9 

Mayer, 92, 93, 94, 96, 97, HS, li24, 

125, 126; 127 
McClung, 98 
Meldolas blue, 53 
Methyl blue, 72, 121 
Methyl eosin, 79 
Methyl green, 21, 22, 69, 120 
Methyl orange, 21, 36, 111 
Methyl violet, 21, 22, 66 
Methyl violet lOB, 67 
Methylene azure, 47, 48, 88, 89, 114 
Methylene blue, 22, 30, 46, 114, 132 
Methylene blue NN, 51 
Methylene blue 0, 50 
Methylene green, 50, 114 
Methylene violet, 47, 49, 88 
Methylene violet RRA, 56 
Meves. 68 

Michaelis, 35, 38, 111, 112 
Milk, staining of, 132 
Mitochondria, staining of, 33, 35, 43, 

65, 68, 70, 98, 134 
Mitosis, 98 
Moll, 95, 126 
Mucin, staining of, 46, 71 
Miiller, 8, 32, 110, 117, 119 
Muscle fibers, staining of, 65, 92, 98 



Naphthaline pink, 5G 

Naphthaline red, 56 

Naphthamine blue, 41 

Napthamine blue 3BX, 41 

Naphthol blue, 53 

Naphthol orange, 36 

Naphthol red, 37 

Naphthol yellow, 32 

N aphthylamine pink, 56 

Narcein, 37, 111 

Nervous tissue, staining of, 37, 40, 43, 

47, 52, 57, 62, 72, 81, 98 
Neuroglia, staining of, 68 
Neutral gentian (Bensley), 34 
Neutral red, 22, 54, 115 
Neutral violet, 54, 115 
Neutral stains, 76, 77, 79, 87-90 
New blue B, 53 
New f uchsin, 20, 64 
New methylene blue N, 51 
New pink, 82 
New Victoria green, 61 
Niagara blue 4B, 41 
Niagara blue 3B, 41 
Niagara sky blue, 41 
Night blue, 71 

Nigrosin, water sol., 22, 57, 116 
Nile blue A, 52 

Nile blue sulphate, 22, 52, 115 
Nissl granules, staining of, 54 



149 



Xitro dyes, 21, 32 

radical, 18 
Nocht, 88, 89 
Nopalin G, 80 

Oil red, 38 

Oil red IV. 38 

Oil yelloic, 41 

Orange I, 36 

Orange II, 37 

Orange III, 30 

Orange IV, 36 

Orange A, P, or R, 37 

Orange extra, 37 

Orange G, 21, 28, 34, 110, 136 

Orange GO, or GMP, 34 

Orange N, 36 

Orange 7?, 41 

Orcein, 94, 126 

Ortho-sulpho-benzoic acid, 83 

Oxazins, 22, 51-53 

Oxyphile granules, 79 

Oxyquinone dyes, 22, 42 

Paladino, 39, 112 

Pancreatic tissue, staining of, 57 

Pappenheim, 50, 70, 76, 90, 115, 120, 
121, 135 

Para-fuchsin, 63 

Para-magenta, 63 

Pararosanilin, 18, 20, 60, 63 

Pararosolic acid, 74 

Paris blue, 71 

Paris violet, 66 

Partsch, 92, 123 

Pelagetti, 112, 123 

Perkin, 9 

Petroff, 61, 117 

Pfitzer, 57, 116 

Phenazin, 17, 5t} 

Phenol, 15 

Phenol red, 84 

Phenolphthalein, 22, 83-4 

Pheneylene blue, 53 

Phenylene brown, 39 

Phenyl-methane dyes, 22, 58-74 

Phloxine, 82, 122 ' 

Phthalic acid, 83 

Phthalic anhydride, 77, 83 

Pianese, 32, 61, Q5, 110, 117, 118 

Picric acid, 9, 15, 21, 32, 110 

I'icro-carmin, 9 

Pith, staining of, 65 

Plants, fungus diseases of, 33, 61, 65 

staining sections of, 60 
• Plgsma fibrils in epithelium, 94 

Platelets, staining of, 67 • 

Ponceau B^ 39 
. Ponceau 3B, 38 

Ponceau 6t{, 41 

Protein, staining of, 56, 70 



Protozoa, staining of, 70 

Pseudo-base, 19 

Purpurin, 43, 113 

Pyoktanin blue, 66 

Pyoktanin yellow, 60 

Pyoktatiimum aureum, 00 

Py renin B or G, 22, 76, 121, 135, 136 

Pyronins, 22, 75-6 

Pyrosin B and ./, 81 

Quinoid ring, 13, 17, 18, 42, 45, 59. 65, 

73, 84 
Quinone, 13 
Quinone-imide dyes, 22, 44-57 

Ranvier, 9, 124 

Rawitz, 94, 125 

Red violet, 65 

Renter, 89 

Rhodamine B, 22, 77, 121 

Rhodaminc 0, 77 

Rhodamine S, 77 

Rhodamines, 22, 77-8 

Rosanilin, 20, 63 

Rosanilins, 62-73 

Robertson, 115 

Roccellin, 41 

Romanovskv, 79, 87, 88, 89, 114, 122 

Rose Bengal, 22, 83, 123 

Rosen, 77 

Rosolic acids, 22, 73-74 

Rothberger, 115 

Rubin, 20, 03 

Rnbidin, 41 

Safranin O, 22, 55, 115, 133 

Safranins, 17, 22, 54-57 

Safrosin, 80 

Salamander larvae, staining of, 60 

Salicin black, 41 

Scarlet B, or FX\ 39 

Scarlet G, or B, 38 

Scarlet J, J J or V, 80 

Scarlet red, 38 

Schaffer, 36, 40, 111, 113 

Scharlach B, 41 

Scharlach R, 38 

Schneider, 93, 124 

Scott, 47, 48 

Schr otter, 43, 113 

Schultz, 127 

Schultz' Index, 23, 106, 107, 109 

Schwa rz, 9 

Schweigger-Seidel, 8 

Silver Gray, 57 

Skin, staining sections of, 36 

Smith, Lorrain, 52, 115 

Smith, Louise, 51 

Society of Dyers and C'olourists, 23 

Solid green, 61 



150 



Soluble blue 3M or 2R. 72 
Spectrophotometer^ 26 

diagram of, 27 
Spectrum, 25 

diagram of, 26 
Spermatozoa, staining of, 62 
Spirit blue, 71 
Spore coats, staining of, 56 
Spuler, 92, 123 
Sputum, staining of, 79 
Steel gray, 51 
Strobell, 112 
Stroebe, 73, 121 
Succineins, 77 
Sudan III, 21, 34, 38, 112 
Sudan IV, 21, 38, 112 
Sudan S, 41 
Sudan G, 38 
Suda7i red, 56 
Sulfonic dyes, 16 
Sulphonphthaleins, 22, 84-5 
Sultan J,B, 40 
Swiss blue, 46 

Tadpoles, vital staining of. 

Teichmiiller, 79, 122 

Thiazins, 22, 44-51 

Thiersch, 8 

Thionin, 22, 30, 31, 45, 113, 137 

Thymol blue, 85 

Tony red, 38 

1'oluene, 14, 58 

Toluidinc, 14 

Toluldine blue 0, 50 

Toluylene blue, 44 

''Triacid mixture" (Khrlich), 34, 87, 88 

Tri-j)henyl methane dyes, 22, 60-74 

Triple stain (Flemming), 34, 5(y, 67, 

102, 110, 119, 135, 136 
Trinitrobenzene, 15 
TropaeoUn D, G, 00. and 000, 36 
Tropaeolin 000 Xo. ?, 37 
Tropaeolin 0, 41 




Tropaeolin Y, 41 
Trypan blue, 41, 113 
Trypan red, 40, 113 

Tubercle organism, staining of, 62 
Tumor tissue, staining of, 46, 52, 98 
Typhoid organism, staining of, 61, 62 

Unna, 36, 39, 54, 57, (i6, 70, 73, 78, 
82,89,94, 103, 104, 111, 115, 116, 
121,122, 126 

Van Gieson, 32, 65, 98, 110, 118, 128 

A'an Wijhe, 51, 124 

Vaughan, 110, 117, 119 

Vesuvin, 39 

Victoria rubin, 37 

Vinassa, 60. 117 

Violet C, G, or 7B, OT 

Violet R, RR, or JfRX, lio 

Vital staining, 35, J^7, 52, 54, 60 

Waldeyer, 9 

Water blue, 72 

Wafer soluble eosin, 79 

Weigert, 67, 98, 118, 119, 125, 127, 128 

V. Wellheim, 125 

Williams, 52, 115 

Winogradsky, 81, 122 

Wood, staining of, 50, 56, 68 

Wool orange 2G, 34 

Wool red, 37 

Wright stain, 87, 89, 136 

Xantheue dyes, 22, 75-85 
Xylem, staining of, 70, 71 
Xylene, 14 

Zacharias, 125 

Ziehl-Neelson method, 62, 118. 133 
Zimmermann. 71, 120 
Zschokke, 41, 113 



151